JP5778577B2 - Antibodies against DLL4 and uses thereof - Google Patents

Description

  The present invention relates to targeted binding agents for DLL4 and the use of such agents. More specifically, the present invention relates to fully human monoclonal antibodies directed against DLL4. These targeted binding agents described are useful in the treatment of diseases associated with DLL4 activity and / or overproduction and as a diagnosis.

  The Notch signaling cascade is an evolutionarily conserved pathway that has been implicated in cell fate decisions, stem cell maintenance and differentiation in many developing tissues. So far, four Notch receptor ligands (Notch1-4) and five ligands (Jagged-1 / 2 and delta-like ligand (Dll) 1/3/4) have been identified in mammals. The Notch receptor exists as a heterodimer and consists of two non-covalently bound extracellular and transmembrane subunits. Ligand binding to extracellular subunits causes protein cleavage by enzymes such as TNFα converting enzyme (TACE) and γ-secretase, resulting in the generation of Notch intracellular region (NICD), which translocates to the nucleus and transcribes It binds to the factor and ultimately leads to activation of the downstream target gene (see, Ray, 2006, Nat. Rev. Mol. Cell. Biol., 7, 678).

  New evidence suggests that many Notch pathway components are expressed in vascular structures and that normal Notch signaling abnormalities can occur in the vascular phenotype. For example, mutations in Jagged 1 and Notch 3 each result in Alagille syndrome, and autosomal dominant cerebral arteropathy with subcortical infarction and leukoencephalopathy, two diseases exhibiting vascular disorders. Furthermore, mouse Notch1 and DLL4 gene deletions all lead to embryonic lethality with vascular abnormalities. Furthermore, deletion of one allele of mouse DLL4 results in embryonic lethality with severe vascular damage in most genetic backgrounds (Duarte et al., 2004, Genes Dev., 18, 2474; Gale et al., 2004, Proc. Nat. Acad. Sci., 101, 15949). This phenotype has only been previously reported for VEGF-A, suggesting that DLL4 may play an important role in vascular development.

  DLL4 expression has also been reported in the vasculature of human renal clear cell carcinoma, glioblastoma, and breast and bladder cancer tumors (Mailhos et al., 2001, Differentiation, 69, 135; Pa vasculature tel et al., 2005). , Cancer Res., 65, 8690; Patel et al., 2006, Clin. Cancer Res., 12, 4836; Li et al., 2007, Cancer Res., 67, 11244). Recent studies also suggested that DLL4 may be expressed in some of the glioblastoma tumor cells (Li et al., 2007, Cancer Res., 67, 11244). The effect of blocking DLL4 signaling on tumor growth was also evaluated using several preclinical models of cancer in which tumor cell lines grow subcutaneously in immunodeficient mice (Ridgway et al., 2006, Nature, 444, 1083; Noguera-Troise et al., Nature, 444, 1032). In these studies, a decrease in tumor growth was reported. These anti-tumor effects were associated with an increase in poorly functioning vascular density in the tumor, concomitant with the worsening of hypoxia. Taken together, these data suggest that in addition to a role in vascular development, DLL4 may also play a role in the development of tumor vasculature.

  In addition to its effect on angiogenesis, Notch signaling is also involved in cancer stem cells from a number of tumor types (Dontu et al., 2004, Breast Can. Res., 6, R605; Wilson & Radtke, 2006, FEBS). Lett., 580, 2860). Cancer stem cells have been isolated from various hematopoietic tumors and solid cancers (Al-Hajj et al., 2003, Proc. Nat. Acad. Sci., 100, 3983; Rapidot et al., 1994, Nature, 17,645). Tan et al., 2006, Laboratory Investigation, 86, 1203), the presence of DLL4 in a small population of tumor cells indicates that DLL4 may also be involved in cancer stem cell biology, and that DLL4 antagonists are associated with these cell types. It further suggests that the anti-tumor effect may be partially mediated through these interactions.

Bray, 2006, Nat. Rev. Mol. Cell. Biol., 7, 678 Duarte et al., 2004, Genes Dev., 18, 2474 Gale et al., 2004, Proc. Nat. Acad. Sci., 101, 15949 Mailhos et al., 2001, Differentiation, 69,135 Pa vasculature tel et al., 2005, Cancer Res., 65, 8690 Patel et al., 2006, Clin. Cancer Res., 12, 4836 Li et al., 2007, Cancer Res., 67, 11244 Ridgway et al., 2006, Nature, 444, 1083 Noguera-Troise et al., Nature, 444, 1032. Dontu et al., 2004, Breast Can. Res., 6, R605 Wilson & Radtke, 2006, FEBS Lett., 580, 2860 Al-Hajj et al., 2003, Proc. Nat. Acad. Sci., 100, 3983 Lapidot et al., 1994, Nature, 17,645. Tan et al., 2006, Laboratory Investigation, 86, 1203.

  The present invention relates to a targeted binding agent that specifically binds to DLL4 and inhibits the biological activity of DLL4. Embodiments of the invention relate to targeted binding agents that specifically bind to DLL4 and inhibit binding of DLL4 to a Notch receptor (eg, Notch 1, 2, 3, or 4).

  Embodiments of the invention relate to targeted binding agents that specifically bind to DLL4 and inhibit binding of DLL4 to a Notch receptor, such as Notch1 or 4. In one embodiment, the targeted binding agent specifically binds DLL4 and inhibits binding to a Notch receptor, such as Notch1. In one embodiment, the targeted binding agent has at least 5%, at least 10%, at least 15 binding of DLL4 to a Notch receptor (eg, Notch1) as compared to binding that occurs in the absence of the targeted binding agent. %, At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, Inhibits at least 80%, at least 85%, at least 90%, at least 95%.

In some embodiments of the invention, the targeted binding agent binds DLL4 with a binding affinity (K _D ) of less than 5 nanomolar (nM). In other embodiments, the targeted binding agent, 4 nM, 3 nM, bind with a _{K D} of less than 2nM or 1 nM. In some embodiments of the present invention, the targeted binding agent binds with DLL4 with a _{K D} of less than 950 picomolar (pM). In some embodiments of the present invention, the targeted binding agent binds with DLL4 with a _{K D} of less than 900 pM. In other embodiments, the targeted binding agent, 800 pM, bind with a _{K D} of less than 700pM or 600 pM. In some embodiments of the present invention, the targeted binding agent binds with DLL4 with a _{K D} of less than 500 pM. In other embodiments, the targeted binding agent binds with a _{K D} of less than 400 pM. In still other embodiments, the targeted binding agent binds with a _{K D} of less than 300 pM. In some other embodiments, the targeted binding agent binds with a K _D of less than 200 pM. In some other embodiments, the targeted binding agent binds with a K _D of less than 150 pM. In yet another embodiment, the targeted binding agent binds with a _{K D} of less than 100 pM. In another embodiment, the targeted binding agent binds with a _{K D} of less than 50 pM. In one specific embodiment, the targeted binding agent of the invention can bind human DLL4 with an affinity K _D of less than 10 pM. In another specific embodiment, the targeted binding agent of the invention can bind human DLL4 with an affinity K _D of less than 1 pM. _The K _D, methods known in the methods or those skilled in the art described herein (e.g., BIAcore assay, ELISA, FACS) (Biacore International AB, Uppsala, Sweden) can be assessed using.

The binding properties of a targeted binding agent or antibody of the invention can also be measured by reference to the dissociation rate or association rate (k _off and k _on , respectively).

In one embodiment of the invention, the targeted binding agent or antibody is at least 10 ⁴ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁴ M ⁻¹ s ⁻¹ , at least 10 ⁵ M ⁻¹ s ⁻¹ , at least 2 ×. 10 ⁵ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁵ M ⁻¹ s ⁻¹ , at least 10 ⁶ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁶ M ⁻¹ s ⁻¹ , at least 10 ⁷ M ⁻¹ s ^-1, may have a _{k on} rate of at least 5 × ¹⁰ 7 ^{M -1 s} -1, or at least ¹⁰ 8 ^M -1 ^{s -1,} (antibody (Ab) + antigen ^{(Ag) kon → Ab-Ag} ).

In another embodiment of the invention, the targeted binding agent or antibody is less than 5 × 10 ⁻¹ s ^−1, less than 10 ⁻¹ s ^−1, less than 5 × 10 ⁻² s ⁻¹ , 10 ⁻² s ^−1. Less than 5 × 10 ⁻³ s ^−1, less than 10 ⁻³ s ^−1, less than 5 × 10 ⁻⁴ s ^−1, less than 4 × 10 ⁻⁴ s ^−1, less than 3 × 10 ⁻⁴ s ⁻¹ , Less than 2 × 10 ⁻⁴ s ^−1, less than 10 ⁻⁴ s ^−1, less than 5 × 10 ⁻⁵ s ^−1, less than 10 ⁻⁵ s ^−1, less than 5 × 10 ⁻⁶ s ⁻¹ , and 10 ⁻⁶ s Less than ^−1, less than 5 × 10 ⁻⁷ s ^−1, less than 10 ⁻⁷ s ^−1, less than 5 × 10 ⁻⁸ s ^−1, less than 10 ⁻⁸ s ^−1, less than 5 × 10 ⁻⁹ s ⁻¹ , It can have a k _off rate ((Ab−Ag) ^koff → antibody (Ab) + antigen (Ag)) of less than 10 ⁻⁹ s ⁻¹ or less than 10 ⁻¹⁰ s ⁻¹ .

The targeted binding agent of the present invention binds to human DLL4. In some examples, the targeted binding agents of the invention are cross-reactive with other DLL4 proteins from other species. In one embodiment, the targeted binding agent of the invention is cross-reactive with cynomolgus DLL4. In another embodiment, the targeted binding agent of the invention exhibits cross-reactivity with cynomolgus monkey DLL4, but exhibits slight cross-reactivity with DLL4 proteins from other species, such as mouse DLL4, for example as assessed by BIAcore technology, it binds mouse DLL with a _{K D} greater than 360 nM.

  In another embodiment, the targeted binding agent of the invention has approximately equivalent affinity for DLL4 proteins from other species. In one embodiment, the human DLL4 targeted binding agent of the invention has approximately equivalent affinity for cynomolgus DLL4. Equivalent level of affinity means that when the affinity for human DLL4 is 1, the affinity of the antibody for cynomolgus DLL4 is 0.2-5 or 0.2-2.

  In another embodiment, the targeted binding agent of the invention is specific for human DLL4 but does not bind to other Notch ligands. In one example, the targeted binding agent of the invention is specific for human DLL4 but does not significantly bind to DLL1, eg, the DLL1 / mock rate is as described herein for targeted binding agents ( 15-300 μg / ml) as measured by testing the ability to bind to 293T cells transiently transfected with human DLL1 or HEK293 cells stably transfected with DLL1. Is less than. In another example, the targeted binding agent of the present invention is specific for human DLL4 but does not significantly bind to Jagged1; for example, the DLL1 / false rate is as described herein for targeted binding agent (5 μg / Ml) as measured by testing the ability to bind to 293T cells transiently transfected with human Jagged 1 or HEK 293 cells stably transfected with human Jagged 1 is there.

In yet another embodiment, the targeted binding agent of the invention inhibits DLL4-Notch1 receptor-ligand binding. In one example, the activity possessed by the targeted binding agent can be shown at an IC ₅₀ concentration (concentration reaching 50% inhibition) of less than 10 μM. In another example, the targeted binding agent of the invention is 50, 40, 30, 20, 10, 5, 4, 2, 1, 0.8, 0.7, 0.6, 0.5 or 0.4 nM. It may have an IC ₅₀ concentration of less than.

  In yet another embodiment, the targeted binding agent of the invention can have both in vitro and in vivo activity. In one embodiment, the targeted binding agent reverses DLL4-stimulated inhibition of HUVEC cell proliferation in two-dimensional culture. In one example, an antibody of the invention has 70% or more, eg, 75%, 80%, 85%, 90%, or compared to a control (using the assay of Example 9 without the addition of DLL4), or 95%, the inhibition of DLL4 stimulation of HUVEC cell proliferation can be reversed.

  In yet another embodiment, the targeted binding agent, eg, an antibody of the invention, can inhibit HUVEC tube formation in two-dimensional culture. For example, an antibody of the invention has an inhibition of more than 50% at a concentration of 0.08 μg / ml relative to a control (this control is the maximum inhibitory effect measured using 20 μg / ml of the same antibody), eg An inhibition of 50%, 60%, 70%, 80%, 90%, or 95% can be shown.

  In yet another embodiment, the targeted binding agent of the invention is less than 50%, eg, 45%, 40%, 35%, 30%, at 4 hours relative to the control at time 0. An internalization of 25%, 20%, 15%, 10% or 5% can be indicated. In another embodiment, the targeted binding agent of the invention exhibits 5-50%, 10-40% or 20-40% internalization at 4 hours relative to the control at time 0. (See Example 15).

In one embodiment, specifically targeted binding agent of the present invention to bind to DLL4 may exhibit one or more of the following characteristics, in its properties, that binds to human DLL4 with a K _D of less than 200 pM; Cross-reacts with cynomolgus monkey DLL4; weakly cross-reacts with mouse DLL4; binds cynomolgus DLL4 with approximately equal affinity; does not significantly bind to DLL1 or Jagged1; HUVEC cell proliferation in two-dimensional cultures compared to controls Shows more than 85% reversal of inhibition by DLL4 stimulation; shows more than 50% inhibition of HUVEC cell tube formation in two-dimensional cultures at a concentration of 0.08 μg / ml compared to control; and time 0 It includes an internalization of less than 50% at the 4 hour time point relative to the control at time.

  In another embodiment, the target binding proteins disclosed herein possess beneficial and effective metabolic and / or pharmacodynamic properties.

  In another embodiment of the invention, the targeted binding agent is any one fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7 for binding to Notch1. Compete with 4B3, 1D4 or 21H3RK.

  In some embodiments of the invention, the targeted binding agent inhibits mammalian tumor growth and / or metastasis. In another embodiment, the targeted binding agent can treat a disease associated with angiogenesis.

  In some embodiments of the invention, the targeted binding agent is an antibody. In some embodiments of the invention, the targeted binding agent is a monoclonal antibody. In one embodiment of the invention, the targeted binding agent is a fully human monoclonal antibody. In another embodiment of the invention, the targeted binding agent is a fully human monoclonal antibody of the IgG1, IgG2, IgG3 or IgG4 isotype. In another embodiment of the invention, the targeted binding agent is a fully human monoclonal antibody of the IgG2 isotype. IgG2 isotypes reduce the likelihood of eliciting effector functions compared to other isotypes, which may reduce toxicity. In another embodiment of the invention, the targeted binding agent is a fully human monoclonal antibody of the IgG1 isotype. The IgG1 isotype increases the likelihood of eliciting ADCC compared to other isotypes, which may improve efficacy. The IgG1 isotype improves stability compared to other isotypes, eg, IgG4, which can improve bioavailability or improve ease of manufacture or have a longer half-life . In one embodiment, the IgG1 isotype fully human monoclonal antibody is a z, za or f allotype antibody.

  A further embodiment is a targeted binding agent or antibody that specifically binds to DLL4 and comprises a sequence comprising one of the complementarity determining region (CDR) sequences shown in Table 2. Embodiments of the invention include a targeted binding agent or antibody comprising a sequence comprising any one CDR1, CDR2 or CDR3 sequence as shown in Table 2. A further embodiment is a targeted binding agent or antibody that specifically binds to DLL4 and comprises a sequence comprising two CDR sequences as shown in Table 2. In another embodiment, the targeted binding agent or antibody comprises a sequence comprising CDR1, CDR2 and CDR3 sequences as shown in Table 2. In another embodiment, the targeted binding agent or antibody comprises a sequence comprising one of the CDR sequences as shown in Table 2. Embodiments of the invention include a targeted binding agent or antibody comprising a sequence comprising any one CDR1, CDR2 or CDR3 sequence as shown in Table 2. In another embodiment, the targeted binding agent or antibody comprises a sequence comprising two CDR sequences as shown in Table 2. In another embodiment, the targeted binding agent or antibody comprises a sequence comprising CDR1, CDR2 and CDR3 sequences as shown in Table 2. In another embodiment, the targeted binding agent or antibody may comprise a sequence comprising CDR1, CDR2 and CDR3 sequences of a variable heavy chain sequence or variable light chain sequence as shown in Table 2. In some embodiments, the targeted binding agent is an antibody. In certain embodiments, the targeted binding agent is a fully human monoclonal antibody. In certain other embodiments, the targeted binding agent is a binding fragment of a fully human monoclonal antibody.

  In another embodiment, the targeted binding agent has an amino acid sequence identical to VH CDR1 as shown in Table 2 or comprising 1, 2, or 3 amino acid residue substitutions for VH CDR1 in Table 2. VH CDR1 as shown, VH as shown in Table 2 having an amino acid sequence identical to VH CDR2 as shown in Table 2 or comprising 1, 2, or 3 amino acid residue substitutions for VH CDR2 CDR2, VH CDR3 as shown in Table 2, having an amino acid sequence identical to VH CDR3 as shown in Table 2 or having 1, 2, or 3 amino acid residue substitutions for VH CDR3, in Table 2 Table 2 having an amino acid sequence identical to VL CDR1 as shown or comprising 1, 2, or 3 amino acid residue substitutions to VL CDR1 VL CDR1 as shown, VL as shown in Table 2 having an amino acid sequence identical to VL CDR2 as shown in Table 2 or comprising 1, 2, or 3 amino acid residue substitutions for VL CDR2 CDR2, comprising VL CDR3 as shown in Table 2 having an amino acid sequence identical to VL CDR3 as shown in Table 2 or comprising 1, 2, or 3 amino acid residue substitutions for VL CDR3.

  In one embodiment, the targeted binding agent comprises VH CDR1, CDR2 and CDR3 as shown in Table 2 and VL CDR1, CDR2 and CDR3 as shown in Table 2.

  In yet another embodiment, the targeted binding agent immunospecifically binds DLL4 and has a heavy chain variable region as shown in Table 2 having at least 90% identity with an amino acid as shown in Table 2. A light chain variable region as shown in Table 2 having at least 90% identity with an amino acid sequence as shown in Table 2, wherein the antibody has binding activity to DLL4.

In another embodiment, the targeted binding agent is deposited with the American Type Culture Collection (ATCC) on September 17, 2008 under the number PTA-9502, PTA-9517, PTA-9501, or PTA-9508. Among the plasmids designated Mab2H10 VHOP, Mab9G8VH, Mab21H3VH, and Mab4B4VH may include sequences comprising any one of the variable heavy chain sequences CDR1, CDR2 or CDR3 encoded by the polynucleotide. In another embodiment, the targeted binding agent is Mab9G8VLOPT1, deposited with the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9516, PTA-9500, or PTA-9520, Among the Mab21H3VLOP and Mab4B4VL and the plasmid designated Mab2H10VLOP 1 deposited on PTA-10181 on July 7, 2009, among the CDR1, CDR2 or CDR3 of the variable light chain sequence encoded by the polynucleotide It may contain a sequence comprising any one.

  In one embodiment, the targeted binding agent or antibody of the invention is contained in a plasmid designated Mab2H10VHOP deposited with the American Type Culture Collection (ATCC) under the number PTA-9502 on September 17, 2008. Includes a variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide.

In one embodiment, the targeted binding agent or antibody of the invention is contained in a plasmid designated Mab2H10VHOP deposited with the American Type Culture Collection (ATCC) under the number PTA-9502 on September 17, 2008. Includes the variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide and is named Mab2H10VLOP 1 deposited on July 7, 2009, with the number PTA-10181, and deposited with the American Type Culture Collection (ATCC) The variable plasmid comprises a variable light chain amino acid sequence comprising CDR3 encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VHOP deposited at the American Type Culture Collection (ATCC) numbered PTA-9502 on September 17, 2008. Including a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VLOP 1 deposited with the American Type Culture Collection (ATCC) on July 7, 2009, numbered PTA-10181. Among them, a variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VHOP deposited at the American Type Culture Collection (ATCC) numbered PTA-9502 on September 17, 2008. A variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide, the American type with the number PTA-10181 on July 7, 2009 A variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide in a plasmid designated Mab2H10VLOP 1 deposited with the Culture Collection (ATCC) including .

  In one embodiment, a targeted binding agent or antibody of the invention is encoded by a polynucleotide in a plasmid designated Mab9G8VH deposited with the American Type Culture Collection (ATCC) under the number PTA-9517. A variable heavy chain amino acid sequence comprising CDR3.

  In one embodiment, the targeted binding agent or antibody of the invention is contained in a plasmid designated Mab9G8VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9517 on September 17, 2008. Includes the variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide and is named Mab9G8VLOPT1, deposited with the American Type Culture Collection (ATCC) under the number PTA-9516 on September 17, 2008. A variable light chain amino acid sequence comprising CDR3 encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9517. Including a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VLOPT1, deposited with the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9516. Including a variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9517. A variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide, the American type with the number PTA-9516 on September 17, 2008 A variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide in a plasmid designated Mab9G8VLOPT1, deposited with the Culture Collection (ATCC) Including.

  In one embodiment, the targeted binding agent or antibody of the invention is contained in a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. Includes a variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide.

  In one embodiment, the targeted binding agent or antibody of the invention is contained in a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. Includes a variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide and is named Mab21H3VLOP deposited with the American Type Culture Collection (ATCC) under the number PTA-9500 on September 17, 2008. A variable light chain amino acid sequence comprising CDR3 encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the present invention comprises a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. Including a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid named Mab21H3VLOP deposited with the American Type Culture Collection (ATCC) under the number PTA-9500 on September 17, 2008. Including a variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide.

  In another embodiment, the targeted binding agent or antibody of the present invention comprises a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. A variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide, the American type with the number PTA-9500 on September 17, 2008 A variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide in a plasmid designated Mab21H3VLOP deposited with the Culture Collection (ATCC) Including.

  In one embodiment, the targeted binding agent or antibody of the present invention is in a plasmid designated Mab4B4VH deposited at the American Type Culture Collection (ATCC) numbered PTA-9508 on September 17, 2008. A variable heavy chain amino acid sequence comprising CDR3 encoded by

  In one embodiment, the targeted binding agent or antibody of the present invention is in a plasmid designated Mab4B4VH deposited at the American Type Culture Collection (ATCC) numbered PTA-9508 on September 17, 2008. A variable heavy chain amino acid sequence comprising CDR3 encoded by the polynucleotide of and named Mab4B4VL deposited at the American Type Culture Collection (ATCC) under the number PTA-9520 on September 17, 2008 It includes a variable light chain amino acid sequence comprising CDR3 encoded by a polynucleotide in a plasmid.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid named Mab4B4VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9508. A variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab4B4VL deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9520. A variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid named Mab4B4VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9508. Comprising a variable heavy chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by a polynucleotide therein, numbered PTA-9520 on September 17, 2008 as an American type A variable light chain amino acid sequence comprising at least one, at least two, or at least three CDRs of an antibody encoded by a polynucleotide in a plasmid named Mab4B4VL deposited with the Culture Collection (ATCC).

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VHOP deposited at the American Type Culture Collection (ATCC) numbered PTA-9502 on September 17, 2008. The variable heavy chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9517. The variable heavy chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the present invention comprises a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. The variable heavy chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid named Mab4B4VH deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9508. The variable heavy chain of the antibody encoded by the polynucleotide therein.

In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VLOP 1 deposited with the American Type Culture Collection (ATCC) on July 7, 2009, numbered PTA-10181. The variable light chain of the antibody encoded by the polynucleotide in

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VLOPT1, deposited with the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9516. The variable light chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid named Mab21H3VLOP deposited with the American Type Culture Collection (ATCC) under the number PTA-9500 on September 17, 2008. The variable light chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab4B4VL deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9520. The variable light chain of the antibody encoded by the polynucleotide therein.

In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab2H10VHOP deposited at the American Type Culture Collection (ATCC) numbered PTA-9502 on September 17, 2008. A plasmid designated as Mab2H10VLOP 1 deposited on July 7, 2009 with the American Type Culture Collection (ATCC), containing the variable heavy chain of the antibody encoded by the polynucleotide therein The variable light chain of the antibody encoded by the polynucleotide in

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab9G8VLOPT1, deposited with the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9516. Of a plasmid named Mab9G8VH deposited with the American Type Culture Collection (ATCC) under the number PTA-9517 on September 17, 2008, including the variable light chain of the antibody encoded by The variable heavy chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the present invention comprises a plasmid designated Mab21H3VH deposited at the American Type Culture Collection (ATCC) under the number PTA-9501 on September 17, 2008. Of a plasmid named Mab21H3VLOP deposited with the American Type Culture Collection (ATCC) numbered PTA-9500 on September 17, 2008, including the variable heavy chain of the antibody encoded by The variable light chain of the antibody encoded by the polynucleotide therein.

  In another embodiment, the targeted binding agent or antibody of the invention is a plasmid designated Mab4B4VL deposited at the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9520. Of a plasmid named Mab4B4VL deposited with the American Type Culture Collection (ATCC) on September 17, 2008, numbered PTA-9520, containing the variable light chain of the antibody encoded by The variable light chain of the antibody encoded by the polynucleotide therein.

  Note that one skilled in the art can easily make CDR determinations. For example, see Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. Kabat provides multiple sequence alignments of immunoglobulin chains from a number of species antibody isotypes. The aligned sequences are numbered according to the Kabat numbering system, which is a single numbering system. The Kabat sequence has been updated since its publication in 1991 and can be used as an electronic sequence database (latest downloadable version is 1997). Any immunoglobulin sequence can be numbered according to Kabat by alignment with the Kabat reference sequence. Thus, Kabat numbering provides a unified system for immunoglobulin chain numbering.

  In one embodiment, the targeted binding agent or antibody comprises a sequence comprising any one heavy chain sequence shown in Table 2. In another embodiment, the targeted binding agent and antibody are any one of the antibodies 20G8, 21H3, 14B1, 18B7, 17B6, 17F3, 12A1, 17G12, 19G9, 21F7, 20D6, 1D4, 4B4, 2H10 or 21H3RK. It includes any optimal variation of sequences comprising chain sequences or heavy chain sequences of these antibodies as shown in Table 5, Table 7, Table 9, Table 11 or Table 13. The mixing of light chains is well established in the art and thus targeted binding agents or antibodies are antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4. Or a sequence comprising any one light chain sequence of 21H3RK, or any optimal variant of the light chain sequences of these antibodies as shown in Table 6, Table 8, Table 10, Table 12 or Table 13, Or any other antibody disclosed herein. In one embodiment, the targeted binding agent or antibody is any one heavy chain sequence as shown in Table 2, or these antibodies as shown in Table 5, Table 7, Table 9, Table 11 or Table 13 Any one of the light chain sequences shown in Table 6, Table 8, Table 10, Table 12, or Table 13, or any of the heavy chain sequences of Any further antibody may further be included. In some embodiments, the antibody is a fully human monoclonal antibody.

  In some embodiments, the targeted binding agent is a monoclonal antibody selected from the group consisting of 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. . In one embodiment, the targeted binding agent comprises one or more fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. In certain embodiments, the targeted binding agent is monoclonal antibody 4B4. In certain embodiments, the targeted binding agent is monoclonal antibody 21H3. In certain embodiments, the targeted binding agent is monoclonal antibody 2H10. In certain embodiments, the targeted binding agent is monoclonal antibody 9G8. In certain other embodiments, the targeted binding agent is monoclonal antibody 21H3RK.

  In one embodiment, the targeted binding agent or antibody may comprise a sequence comprising heavy chains CDR1, CDR2 and CDR3 selected from any one of the sequences shown in Table 2.

  In one embodiment, the targeted binding agent or antibody may comprise a sequence comprising light chain CDR1, CDR2 and CDR3 selected from any one sequence shown in Table 2. In one embodiment, the targeted binding agent or antibody is a heavy selected from any one CDR of antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. It may comprise a sequence comprising chains CDR1, CDR2 and CDR3. In one embodiment, the targeted binding agent or antibody is a light chain selected from any one CDR of antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4 or 21H3RK. It may include sequences comprising CDR1, CDR2 and CDR3.

  In another embodiment, the targeted binding agent or antibody is any one of the variable heavy chain sequences of any one fully human monoclonal antibody 4B4, as shown in Table 2, or 21H3, 2H10, 9G8, or 21H3RK. A sequence comprising one CDR1, CDR2 or CDR3 may be included. In another embodiment, the targeted binding agent or antibody is any one of the fully light monoclonal antibodies 4B4 as shown in Table 2, or any of the variable light chain sequences of 21H3, 2H10, 9G8, or 21H3RK. A sequence comprising one CDR1, CDR2 or CDR3 may be included. In another embodiment, the targeted binding agent or antibody is a fully human monoclonal antibody 4B4 as shown in Table 2, or a sequence comprising CDR1, CDR2 and CDR3 of 21H3, 2H10, 9G8, or 21H3RK, and The fully human monoclonal antibody 4B4 as shown, or may contain 21H3, 2H10, 9G8, or 21H3RK CDR1, CDR2 and CDR3 sequences. In some embodiments, the antibody is a fully human monoclonal antibody.

  In another embodiment, the targeted binding agent or antibody comprises a sequence comprising the CDR1, CDR2 and CDR3 sequences of fully human monoclonal antibody 4B4 as shown in Table 2, and the CDR1, CDR2 and CDR3 sequences of fully human monoclonal antibody 4B4. including. In another embodiment, the targeted binding agent or antibody is a sequence comprising CDR1, CDR2 and CDR3 sequences of fully human monoclonal antibody 21H3 as shown in Table 2, and fully human monoclonal antibody 21H3 as shown in Table 2. CDR1, CDR2 and CDR3 sequences. In another embodiment, the targeted binding agent or antibody is a fully human monoclonal antibody 2H10 sequence as shown in Table 2, including the CDR1, CDR2 and CDR3 sequences of fully human monoclonal antibody 2H10, and a fully human monoclonal antibody 2H10 as shown in Table 2. CDR1, CDR2 and CDR3 sequences. In some embodiments, the antibody is a fully human monoclonal antibody.

  Further embodiments of the invention include sequences comprising any one sequence framework region as shown in Table 2 or Table 13 and contiguous sequences spanning CDRs, specifically FR1-FR4 or CDR1-CDR3. Targeted binding agent or antibody. In one embodiment, the targeted binding agent or antibody is a monoclonal antibody 4B4 as shown in Table 2 or Table 13, or a framework region and CDR of any one sequence of 21H3, 2H10, 9G8, or 21H3RK, specifically Specifically, it includes a sequence including a contiguous sequence spanning FR1 to FR4 or CDR1 to CDR3. In some embodiments, the antibody is a fully human monoclonal antibody.

  In another embodiment, the agent or antibody, or antigen binding portion thereof, comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO: 2. In one embodiment, the agent or antibody, or antigen binding portion thereof, further comprises a light chain polypeptide comprising the sequence of SEQ ID NO: 4. In some embodiments, the antibody is a fully human monoclonal antibody.

  One embodiment provides a targeted binding agent or antibody, or antigen-binding portion thereof, comprising a heavy chain polypeptide comprising the sequence of SEQ ID NO: 6. In one embodiment, the agent or antibody, or antigen binding portion thereof, further comprises a light chain polypeptide comprising the sequence of SEQ ID NO: 8. In some embodiments, the antibody is a fully human monoclonal antibody.

  In another embodiment, the agent or antibody, or antigen binding portion thereof, comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO: 22. In another embodiment, the agent or antibody, or antigen binding portion thereof, further comprises a light chain polypeptide comprising the sequence of SEQ ID NO: 24. In some embodiments, the antibody is a fully human monoclonal antibody.

  In another embodiment, the agent or antibody, or antigen binding portion thereof, comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO: 30. In another embodiment, the agent or antibody, or antigen binding portion thereof, further comprises a light chain polypeptide comprising the sequence of SEQ ID NO: 32. In some embodiments, the antibody is a fully human monoclonal antibody.

  In another embodiment, the agent or antibody, or antigen binding portion thereof, comprises a heavy chain polypeptide comprising the sequence of SEQ ID NO: 30. In another embodiment, the agent or antibody, or antigen binding portion thereof, further comprises a light chain polypeptide comprising the sequence of SEQ ID NO: 50. In some embodiments, the antibody is a fully human monoclonal antibody.

  In one embodiment, the targeted binding agent or antibody is 20, 16, 10, 9 or less, eg 1, 2, 3, 4, or 5 in the disclosed CDR or heavy or light chain sequence. Of amino acid additions, substitutions, deletions, and / or insertions. Such modifications can potentially occur at any residue in the CDR. In some embodiments, the antibody is a fully human monoclonal antibody.

  In one embodiment, the targeted binding agent or antibody comprises a variant or derivative of a CDR disclosed herein, a framework region and a contiguous sequence spanning a CDR (specifically FR1-FR4 or CDR1-CDR3), A light chain or heavy chain sequence disclosed in the specification, or an antibody disclosed herein. Variants are no more than 20, 16, 10, 9, eg, 1, 2, 3, 4, 5 or 6 amino acids in any CDR1, CDR2 or CDR3 as shown in Table 2 or Table 13. Sequences having additions, substitutions, deletions, and / or insertions, framework regions as shown in Table 2 or Table 13, and contiguous sequences spanning CDRs (specifically FR1-FR4 or CDR1-CDR3), A targeted binding agent or antibody comprising a light or heavy chain sequence disclosed herein or a monoclonal antibody disclosed herein. A variant is a sequence having at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with any CDR1, CDR2 or CDR3 as shown in Table 2 or Table 13, Table 2 or contiguous sequences spanning framework regions and CDRs (specifically FR1-FR4 or CDR1-CDR3) as shown in Table 13, light chain or heavy chain sequences disclosed herein, or A targeted binding agent or antibody comprising the monoclonal antibody disclosed in. The percent identity between two amino acid sequences can be determined by any method known to those of skill in the art, including but not limited to pairwise protein alignments. In one embodiment, the variant is a CDR sequence or light or heavy chain disclosed herein that is naturally occurring or introduced by in vitro manipulation of the native sequence using recombinant DNA or mutagenesis methods. Changes are included in the polypeptide. Naturally occurring variants include those that are generated in vivo in the corresponding germline nucleotide sequence during the production of antibodies against foreign antigens. In one embodiment, the derivative can be a heterologous antibody that is an antibody in which two or more antibodies are linked together. Derivatives include chemically modified antibodies. Examples include covalent bonding of one or more polymers, such as water soluble polymers, N-linked or O-linked carbohydrates, sugars, phosphates, and / or other such molecules. Derivatives are modified in a different manner than naturally occurring or starting antibodies, either in the type or position of the molecule to be bound. Derivatives further include deletion of one or more chemical groups that are naturally present on the antibody.

  In one embodiment, the targeted binding agent is a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Methods for making bispecific antibodies are known in the art (eg, Millstein et al, Nature, 305: 537-539 (1983); Traunecker et al, EMBO J, 10: 3655-3659 ( 1991); Suresh et al, Methods in Enzymology, 121: 210 (1986); Kostelny et al, J. Immunol, 148 (5): 1547-1553 (1992); Hollinger et al, Proc. Natl Acad. 90: 6444-6448 (1993); Gruber et al, J. Immunol, 152: 5368 (1994); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925, U.S. Pat. 648 5,573,920; 5,601,81; 95,731,168; 4,676,980; and 4,676,980; WO94 / 04690 WO 91/00360; WO 92/003733; WO 93/17715; WO 92/08802; and European Patent No. 03089).

  In some embodiments of the invention, the targeted binding agent or antibody has its sequence optimized by mutating non-germline residues to germline residues and / or removing structural disturbances. Have. In one embodiment, the invention comprises a sequence comprising SEQ ID NO: 6. In certain embodiments, SEQ ID NO: 6 comprises any one combination of germline and non-germline residues shown in each row of Table 7. In some embodiments, SEQ ID NO: 6 is any one, any two, any three, any four, any five, any six, or any six as shown in Table 7. Contains all 6 germline residues. In certain embodiments, SEQ ID NO: 6 comprises any one unique combination of germline and non-germline residues shown in each row of Table 7.

  In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having VH3-33, 6-13, and JH4 regions, wherein one or more residues correspond to that position in the germline. Mutates to yield residues. In certain embodiments, SEQ ID NO: 24 can further comprise a modification that includes removing a structural hindrance. For example, in addition to the reproductive system, C33 can be mutated to S. Thus, SEQ ID NO: 24 can include any one unique combination of germline and non-germline residues shown in each row of Table 10, and can further include a C33 to S mutation.

  A further embodiment of the invention is a targeted binding agent or antibody that competes for binding to DLL4. In another embodiment of the invention, the invention is directed to a targeted binding agent or antibody that competes with native DLL4 for binding to the Notch 1 or Notch 4 receptor. In another embodiment, the targeted binding agent or antibody competes for binding to Notch 1 with any one fully human monoclonal antibody 4B4, or 21H3, 2H10, 9G8, or 21H3RK. “Competing” means that this targeted binding agent or antibody is any one fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK and Notch. Compete for binding to 1, indicating that the competition is unidirectional.

  Embodiments of the present invention seek to bind any one fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK to DLL4 Target binding agents or antibodies that compete with each other. “Competing with each other” means that the targeted binding agent or antibody is any one fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or Compete for binding to 21H3RK and Notch 1, and vice versa, indicating that the competition is bidirectional.

  A further embodiment of the invention is a targeted binding agent or antibody that binds to the same epitope on DLL4 as the targeted binding agent or antibody of the invention. Embodiments of the invention bind to the same epitope on DLL4 as any one fully human monoclonal antibody 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK Also included are targeted binding agents or antibodies. It is clear from cross-competition analysis that the antibodies of the invention have different or partially overlapping epitopes. For example, 4B4 cross-competes with 21H3RK and 21H3. Also, 4B4 and 21H3 do not bind to DLL4 under reducing and denaturing conditions, whereas 9G8 and 2H10 bind, suggesting binding to different epitopes.

  In certain embodiments, the epitope comprises at least one extracellular portion of DLL4. At least one specific epitope (e.g., 21H3 or 21H3RK or 4B4) is between amino acids 147-224 of DLL4, i.e., ICSDNYYGDNCSRLCKKRNNDHFFGHYVCQPGNGELCQQPICLSGCHEQNGYCSKPAECLCPGLCLC specific amino acid residues present in the amino acid sequence number 3 Any combination of at least one amino acid sequence having In one embodiment, the epitope is at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino acid residues, at least 70 amino acids Residue, at least 75 amino acid residues, at least 76 amino acid residues, or 77 amino acid residues. In another embodiment, the epitope is present in TGEYCQQPICLSGCH (SEQ ID NO: 91) at amino acids 187-201 of human DLL4. In one embodiment, the epitope is at least 4 amino acid residues of SEQ ID NO: 91, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 13 amino acid residues, at least 14 amino acid residues, or 15 amino acid residues.

  In one embodiment, the present invention includes mouse cross-reactive antibodies among the antibodies disclosed herein. In one embodiment, the variable regions of these antibodies are modified so that these antibodies can bind to mouse DLL4. In general, mouse cross-reactive antibodies have similar properties as the antibodies disclosed herein, eg, can bind to DLL4 and inhibit binding of DLL4 to the Notch receptor. it can. In one embodiment, the variable region of the 21H3RK antibody is modified so that it can bind to mouse DLL4, for example, this heavy chain has the following changes: H31 Asn to Lys, H52a Ala to Pro, H97 Change Val to Thr, H100b Val to Trp, and H100e Glu to Ala (see SEQ ID NO: 84) and this light chain has the following changes: L30 Ser to Asn and L93 Asp to Ser Change (see SEQ ID NO: 85). In another embodiment, the heavy chain has the following changes: Thr of H30 to Ile, Asn of H31 to Met, Ala of H52a to Pro, Val of H100b to Trp, and Glu of H100e to Ala. Changes (see SEQ ID NO: 86) as well as this light chain have the following changes: L93 Asp changed to Ser (see SEQ ID NO: 87). In yet another embodiment, the heavy chain has the following changes: Thr of H30 to Ile, Asn of H31 to His, Val of H100b to Trp, and Glu of H100e to Ala (see SEQ ID NO: 88) ) As well as this light chain with the following changes: L30 Ser to Asn and L93 Asp to Ser (see SEQ ID NO: 89).

  Other embodiments of the invention have an isolated nucleic acid molecule encoding any targeted binding agent or antibody described herein, an isolated nucleic acid molecule encoding a targeted binding agent or antibody described herein. A host cell transformed with a vector or any such nucleic acid molecule is included. Embodiments of the invention include nucleic acid molecules that encode fully human isolated targeted binding agents that specifically bind to DLL4 and inhibit the binding of DLL4 to the Notch receptor. The present invention provides a polynucleotide that hybridizes to a polynucleotide encoding any targeted binding agent or antibody described herein under stringent or low stringency hybridization conditions as defined herein. Including. Embodiments of the invention also include vectors comprising nucleic acid molecules encoding this binding agent. Additional embodiments include host cells comprising a vector containing the nucleic acid molecule.

  As is known in the art, the antibody can advantageously be, for example, a polyclonal antibody, an oligoclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

It will be appreciated that embodiments of the invention are not limited to any particular type of antibody or production method or method of production. In some embodiments of the invention, the targeted binding agent is a binding fragment of a fully human monoclonal antibody. For example, the targeted binding agent can be a full length antibody (eg, having an intact human Fc region) or an antibody binding fragment (eg, Fab, Fab ′ or F (ab ′) ₂ , FV or dAb). Furthermore, these antibodies can be camel binding to DLL4, such as dAb fragments, or single region antibodies such as human single VH or VL regions.

  The embodiments of the invention described herein also provide cells for producing these antibodies. Examples of cells include hybridomas that produce antibodies to DLL4, or recombinant cells such as Chinese hamster ovary (CHO) cells, CHO cell variants (eg, DG44) and NS0 cells. Detailed information on variants of CHO cells can be found in Andersen and Reilly (2004) Current Opinion in Biotechnology 15, 456-462, which is incorporated herein by reference in its entirety. The antibody can be produced from a hybridoma that secretes the antibody or from recombinantly engineered cells that have been transformed or transfected with one or more genes encoding the antibody.

  Furthermore, one embodiment of the present invention is a method for producing an antibody of the present invention by culturing host cells under conditions in which the nucleic acid molecule is expressed and then recovered to produce the antibody of the present invention. is there. Embodiments of the invention comprise an antibody or antibody fragment of the invention comprising a nucleic acid sequence optimized to increase the yield of the antibody or antibody fragment when transfected into a host cell for antibody production It should be recognized that any nucleic acid molecule that encodes is also included.

  Further embodiments herein include a cell expressing human DLL4, an isolated cell membrane comprising human DLL4, purified human DLL4, or a fragment thereof, and / or one or more orthologous sequences or fragments thereof. Methods of producing antibodies that specifically bind to DLL4 and inhibit the biological activity of DLL4 by immunizing a mammal.

  In other embodiments, the invention provides a composition comprising a targeted binding agent or antibody of the invention, or binding fragment thereof, and a pharmaceutically acceptable carrier or diluent.

  Further embodiments of the invention include methods of effectively treating an animal suffering from a proliferative disease, angiogenic disease by administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. . In certain embodiments, the method selects an animal in need of treatment for a tumor, cancer, and / or cell proliferative disorder and provides the animal with a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. Further comprising.

  Further embodiments of the invention include methods of effectively treating an animal suffering from a neoplastic disease by administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. In certain embodiments, the method further comprises selecting an animal in need of treatment for the neoplastic disease and administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4.

  Further embodiments of the invention include methods of effectively treating an animal suffering from a malignant tumor by administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. In certain embodiments, the method further comprises selecting an animal in need of treatment for a malignant tumor and administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4.

  A further embodiment of the present invention provides a method of effectively treating an animal suffering from a disease or condition associated with DLL4 expression by administering to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. Including. In certain embodiments, the method selects an animal in need of treatment for a disease or condition associated with DLL4 expression and administers to the animal a therapeutically effective amount of a targeted binding agent that specifically binds DLL4. In addition.

  Malignant tumors include melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocyte (liver) cancer, thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer, ovarian cancer, Bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, biliary cancer (cholangiocarcinoma), It may be selected from the group consisting of small intestine adenocarcinoma, childhood malignancy and squamous cell carcinoma.

  Treatable proliferative or angiogenic diseases include melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular (liver) cancer, thyroid tumor, gastric (stomach) cancer, Gallbladder cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, ovary, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, It includes neoplastic diseases such as biliary cancer (cholangiocarcinoma), small intestine adenocarcinoma, childhood malignancy, squamous cell carcinoma and leukemia including chronic myeloid leukemia.

  In one embodiment, the present invention is suitable for use in the inhibition of DLL4 in tumor patients that rely solely on, or in part, on DLL4.

  Further embodiments of the invention include the use of a targeted binding agent or antibody of the invention in the preparation of a medicament for the treatment of an animal suffering from a proliferative or angiogenesis related disease. In certain embodiments, the use further comprises selecting an animal in need of treatment for a proliferative or angiogenesis related disease.

  Further embodiments of the invention include the use of a targeted binding agent or antibody of the invention in the preparation of a medicament for the treatment of an animal suffering from a neoplastic disease. In certain embodiments, the use further comprises selecting an animal in need of treatment for a neoplastic disease.

  Further embodiments of the invention include the use of a targeted binding agent or antibody of the invention in the preparation of a medicament for the treatment of an animal suffering from a non-neoplastic disease. In certain embodiments, the use further comprises selecting an animal in need of treatment for a non-neoplastic disease.

  Further embodiments of the invention include the use of a targeted binding agent or antibody of the invention in the preparation of a medicament for the treatment of an animal suffering from a malignant tumor. In certain embodiments, the use further comprises selecting an animal in need of treatment for a malignant tumor.

  Further embodiments of the invention include the use of a targeted binding agent or antibody of the invention in the preparation of a medicament for the treatment of an animal suffering from a disease or condition associated with DLL4 expression. In certain embodiments, the use further comprises selecting an animal in need of treatment for a disease or condition associated with DLL4 expression.

  Further embodiments of the invention include a targeted binding agent or antibody of the invention for use as a medicament for the treatment of an animal suffering from a proliferative or angiogenesis related disease.

  Further embodiments of the invention include a targeted binding agent or antibody of the invention for use as a medicament for the treatment of an animal suffering from a neoplastic disease.

  Further embodiments of the invention include a targeted binding agent or antibody of the invention for use as a medicament for the treatment of an animal suffering from a malignant tumor.

  Further embodiments of the invention include a targeted binding agent or antibody of the invention for use as a medicament for the treatment of an animal suffering from a disease or condition associated with DLL4 expression.

  Further embodiments of the invention include a targeted binding agent or antibody of the invention for use as a medicament for the treatment of an animal suffering from a DLL4-induced disease.

In one embodiment,
Proliferative or angiogenic disease neoplastic disease;
Treatment of a disease or condition associated with malignancy; or DLL4 expression includes managing, ameliorating, or preventing any of the aforementioned diseases or conditions.

  In one embodiment, treatment of neoplastic disease comprises inhibiting tumor growth, delaying tumor growth, reducing tumors, shrinking tumors, extending time to tumor regrowth upon discontinuation of treatment, time to tumor recurrence Includes prolongation and slowing of disease progression.

  In some embodiments of the invention, the animal to be treated is a human.

  In some embodiments of the invention, the targeted binding agent is a fully human monoclonal antibody.

  In some embodiments of the invention, the targeted binding agent is from the group consisting of fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. Selected.

  Embodiments of the invention include conjugates comprising the targeted binding agents and therapeutic agents described herein. In some embodiments of the invention, the therapeutic agent is a toxin. In other embodiments, the therapeutic agent is a radioisotope. In yet other embodiments, the therapeutic agent is a pharmaceutical composition.

  In another embodiment, a method of selectively killing a patient's cancerous cells is provided. The method includes administering a fully human antibody conjugate to the patient. The fully human antibody conjugate includes an antibody and an agent that can bind to DLL4. The drug is either a toxin, a radioisotope, or another substance that kills cancer cells. Thus, this antibody conjugate selectively kills cancer cells.

  In one aspect, a conjugated fully human antibody that specifically binds to DLL4 is provided. When the drug attaches to the antibody and binds to the cell, the drug is delivered to the cell. In one embodiment, the conjugated fully human antibody binds to the extracellular region of DLL4. In another embodiment, the antibody and conjugated toxin are internalized by cells that express DLL4. In another embodiment, the agent is a cytotoxic agent. In another embodiment, the agent is, for example, saporin or an immunoconjugate based on auristatin, Pseudomonas aeruginosa exotoxin, gelonin, lysine, calicheamicin or maytansine. In yet another embodiment, the agent is a radioisotope.

  The targeted binding agents or antibodies of the present invention can be administered alone or in combination with additional antibodies or chemotherapeutic agents or radiation therapy. For example, a monoclonal, oligoclonal or polyclonal mixture of DLL4 antibodies that block cell adhesion, invasion, angiogenesis, or proliferation can be administered in combination with agents that have been shown to inhibit tumor cell growth.

  Another embodiment of the invention includes a method of diagnosing a disease or condition, wherein the antibodies disclosed herein are utilized to detect the amount of DLL4 in a patient or patient sample. In one embodiment, the patient sample is blood or serum or urine. In further embodiments, methods of risk factor identification, disease diagnosis, and disease staging are provided, the methods including identification of DLL4 expression and / or overexpression using anti-DLL4 antibodies. In some embodiments, the method comprises administering to the patient a fully human antibody conjugate that selectively binds to DLL4 on the cell. The antibody conjugate includes an antibody that specifically binds to DLL4 and a label. The method further includes observing the presence of the label in the patient. A label with a higher relative amount indicates a disease with a higher relative risk and a label with a lower relative amount indicates a disease with a lower relative risk. In one embodiment, the label is green fluorescent protein.

  The invention further provides a method of assaying the amount of DLL4 in a patient sample, the method comprising contacting an antibody disclosed herein with a biological sample from a patient, and DLL4 and the antibody in the sample. And detecting the binding amount. In a more specific embodiment, the biological sample is blood, plasma or serum.

  Another embodiment of the present invention provides a method of diagnosing a condition associated with intracellular DLL4 expression by contacting serum or cells with an antibody disclosed herein and then detecting the presence of DLL4. Including. In one embodiment, the condition can be a disease associated with proliferative, angiogenesis, cell adhesion, or invasion, including but not limited to a neoplastic disease.

  In another embodiment, the invention includes an assay kit that detects DLL4 in mammalian tissue, cells, or body fluids to screen for DLL4-related diseases. The kit includes an antibody disclosed herein and a means for indicating the reaction of the antibody with DLL4, if present. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody that binds DLL4 is labeled. In another embodiment, the antibody is an unlabeled primary antibody and the kit further comprises means for detecting the primary antibody. In one embodiment, the means for detecting comprises a labeled secondary antibody that is an anti-immunoglobulin. The antibody can be labeled with a marker selected from the group consisting of fluorescent dyes, enzymes, radionuclides and radiopaque materials.

  In some embodiments, a targeted binding agent or antibody disclosed herein can be modified to bind to complement and increase its ability to participate in complement dependent cytotoxicity (CDC). In other embodiments, the targeted binding agent or antibody can be modified to activate effector cells and increase their ability to participate in antibody-dependent cellular cytotoxicity (ADCC). In still other embodiments, the targeted binding agent or antibody disclosed herein increases the ability to activate effector cells to participate in antibody dependent cellular cytotoxicity (ADCC) and binds to complement. It can be modified to both enhance its ability to participate in complement dependent cytotoxicity (CDC).

  In some embodiments, a targeted binding agent or antibody disclosed herein can be modified to bind to complement and reduce its ability to participate in complement dependent cytotoxicity (CDC). In other embodiments, the targeted binding agent or antibody can be modified to activate effector cells and reduce their ability to participate in antibody dependent cellular cytotoxicity (ADCC). In still other embodiments, the targeted binding agents or antibodies disclosed herein bind to complement, activating effector cells to reduce their ability to participate in antibody-dependent cellular cytotoxicity (ADCC), and binding to complement. Can be modified to both reduce the ability to participate in complement dependent cytotoxicity (CDC).

  In certain embodiments, the half-life of the targeted binding agent or antibody disclosed herein and the half-life of the composition of the invention is at least about 4-7 days. In certain embodiments, the average half-life of the targeted binding agent or antibody disclosed herein and the average half-life of the composition of the present invention is at least about 2-5 days, 3-6 days, 4-7 days 5-8 days, 6-9 days, 7-10 days, 8-11 days, 8-12 days, 9-13 days, 10-14 days, 11-15 days, 12-16 days, 13-17 days 14-18 days, 15-19 days, or 16-20 days. In other embodiments, the average half-life of the targeted binding agent or antibody disclosed herein and the average half-life of the composition of the present invention is at least about 17-21 days, 18-22 days, 19-23 days. 20 to 24 days, 21 to 25 days, 22 to 26 days, 23 to 27 days, 24 to 28 days, 25 to 29 days, or 26 to 30 days. In further embodiments, the half-life of the targeted binding agent or antibody disclosed herein and the half-life of the composition of the invention can be up to about 50 days. In certain embodiments, the half-life of the antibodies and compositions of the invention can be extended by methods known in the art. Such an extension can then reduce the dose and / or frequency of administration of the antibody composition. Antibodies with improved in vivo half-life and methods for preparing these antibodies are disclosed in US Pat. No. 6,277,375, and International Publications WO 98/23289 and WO 97/3461.

  In another embodiment, the present invention provides a product comprising a container. The container comprises a composition comprising a targeted binding agent or antibody disclosed herein and includes, but is not limited to, diseases characterized by expression or overexpression of DLL4, cell adhesion, invasion, angiogenesis, and A package insert or label indicating that the composition can be used to treat a growth related disorder.

  In other embodiments, the invention provides a kit comprising a composition comprising a targeted binding agent or antibody disclosed herein, and instructions for administering the composition to a subject in need of treatment. I will provide a.

  The present invention provides a formulation of a protein comprising a variant Fc region. This is a non-naturally occurring Fc region, eg, an Fc region comprising one or more non-naturally occurring amino acid residues. Fc regions containing amino acid deletions, additions and / or modifications are also included in the variant Fc region of the present invention.

  The serum half-life of a protein containing an Fc region can be increased by increasing the binding affinity of this Fc region for FcRn. In one embodiment, the Fc variant protein has an increased serum half-life compared to a similar molecule.

  In another embodiment, the invention provides that the Fc region is at least one naturally occurring at one or more positions selected from the group consisting of 239, 330 and 332 numbered by the EU index described in Kabat. Fc variants containing non-existing amino acids are provided. In a specific embodiment, the invention provides an Fc variant comprising at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, wherein the Fc region is numbered by the EU index described in Kabat I will provide a. Optionally, this Fc region further comprises an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, numbered by the EU index described in Kabat. May be included. In a specific embodiment, the invention relates to at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, wherein the Fc region is numbered by the EU index described in Kabat, and Kabat. Provided is an Fc variant comprising at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T, and 256E numbered by the described EU index.

  In another embodiment, the present invention relates to at least one naturally occurring at one or more positions selected from the group consisting of 234, 235 and 331, wherein the Fc region is numbered by the EU index described in Kabat. Fc variants containing non-existing amino acids are provided. In a specific embodiment, the invention comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y and 331S, wherein the Fc region is numbered by the EU index described in Kabat. Fc variants are provided. In a more specific embodiment, the Fc variants of the present invention comprise the non-naturally occurring amino acid residues of 234F, 235F, and 331S numbered by the EU index described in Kabat. In another specific embodiment, the Fc variants of the present invention comprise the non-naturally occurring amino acid residues of 234F, 235Y, and 331S numbered by the EU index described in Kabat. Optionally, this Fc region further comprises an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, numbered by the EU index described in Kabat. May be included. In a specific embodiment, the invention comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y and 331S, wherein the Fc region is numbered by the EU index described in Kabat. Provided is an Fc variant selected from the group consisting of 252Y, 254T, and 256E, wherein at least one non-naturally occurring amino acid at one or more positions is numbered by the EU index described in Kabat.

  In another embodiment, the invention provides that the Fc region is at least one naturally occurring at one or more positions selected from the group consisting of 239, 330 and 332 numbered by the EU index described in Kabat. Fc variant protein formulations comprising non-existing amino acids are provided. In a specific embodiment, the invention provides an Fc variant comprising at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, wherein the Fc region is numbered by the EU index described in Kabat A protein formulation is provided. Optionally, this Fc region further comprises an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, numbered by the EU index described in Kabat. May be included. In a specific embodiment, the invention comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, wherein the Fc region is numbered by the EU index described in Kabat. Provided is an Fc variant protein formulation selected from the group consisting of 252Y, 254T, and 256E, wherein at least one non-naturally occurring amino acid at one or more positions is numbered by the EU index described in Kabat.

  In another embodiment, the present invention relates to at least one naturally occurring at one or more positions selected from the group consisting of 234, 235 and 331, wherein the Fc region is numbered by the EU index described in Kabat. Fc variant protein formulations comprising non-existing amino acids are provided. In a specific embodiment, the invention comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y and 331S, wherein the Fc region is numbered by the EU index described in Kabat. Fc variant protein formulations are provided. Optionally, this Fc region further comprises an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, numbered by the EU index described in Kabat. May be included. In a specific embodiment, the invention comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y and 331S, wherein the Fc region is numbered by the EU index described in Kabat. Provided is an Fc variant protein formulation selected from the group consisting of 252Y, 254T, and 256E, wherein at least one non-naturally occurring amino acid at one or more positions is numbered by the EU index described in Kabat .

  Methods for generating non-naturally occurring Fc regions are known in the art. For example, amino acid substitutions and / or deletions can be made by site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methods and Applications ”, Academic Press, San Diego, pp. 177-183 (1990)), as well as cassette-type mutagenesis (Wells et al, Gene 34: 315-323 (1985)). Can be generated by mutagenesis. Preferably, site-directed mutagenesis can be performed by overlap extension PCR (Higuchi, in “PCR Technology: Principles and Applications for DNA Amplification”, Stockton Press, New York, pp. 61-70). The overlap extension PCR technique (said Higuchi) can also be used to introduce any desired mutation (s) into the target sequence (starting DNA). For example, the first stage of PCR in the overlap extension method uses an outer primer (primer 1) and an internal mutagenesis primer (primer 3), and a second outer primer (primer 4) and an inner primer (primer 2). To separately amplify the target sequence and obtain two PCR fragments (fragment A and fragment B). The internal mutagenesis primer (Primer 3) is designed to contain a mismatch that identifies the desired mutation (s) relative to the target sequence. In the second stage of PCR, the products of the first stage of PCR (fragment A and fragment B) are amplified by PCR using two outer primers (primers 1 and 4). The generated full-length PCR fragment (fragment C) is cleaved with a restriction enzyme, and the generated restriction fragment is cloned into an appropriate vector. As a first step in mutagenesis, the starting DNA (eg, encoding an Fc fusion protein, antibody or simply Fc region) is operably cloned into a mutagenesis vector. These primers are designed to reflect the desired amino acid substitution. Other useful methods for generating variant Fc regions are known in the art (eg, US Pat. Nos. 5,624,821, 5,885,573, 5,677,425). No. 6,165,745, No. 6,277,375, No. 5,869,046, No. 6,121,022, No. 5,624,821, No. 5 648,260, 6,528,624, 6,194,551, 6,737,056, 6,821,505, 6,277,375 US Patent Publication No. 2004/0002587 and PCT Publication No. WO94 / 29351, WO99 / 58572, WO00 / 42072, WO02 / 060919, WO04 / 029207, WO04 / 099249. See No. WO04 / 063351).

  In some embodiments of the invention, the glycosylation pattern of the antibodies provided herein is modified to enhance ADCC and CDC effector function. See Shields RL et al, (2002) JBC.277: 26733; Shinkawa T et al, (2003) JBC.278: 3466 and Okaza A et al, (2004) J. Mol. Biol., 336: 1239. . In some embodiments, the Fc variant protein comprises a carbohydrate composition that is covalently linked to one or more engineered glycoforms, ie, molecules comprising the Fc region. Modified glycoforms can be useful for a variety of purposes including, but not limited to, increasing or decreasing effector function. The modified glycoform can be obtained by any method known to those skilled in the art, for example, by using a modified or variant expression system, such as one or more enzymes, such as DIN-acetylglucosaminyltransferase III (GnTI11). By expressing the molecule (s) comprising the Fc region in different organisms or cell lines derived from different organisms, or after expression of the molecule comprising the Fc region (s) It can be produced by modification. Methods for producing modified glycoforms are known in the art and are described in Umana et al, 1999, Nat. Biotechnol 17: 176-180; Davies et al, 20001 Biotechnol Bioeng 74: 288-294; Shields et al. , 2002, J Biol Chem 277: 26733-26740; Shinkawa et al, 2003, J Biol Chem 278: 3466-3473, US Pat. No. 6,602,684; US Patent Application No. 10 / 277,370; Application No. 10 / 113,929; PCTWO00 / 61739A1; PCTWO01 / 292246A1; PCTWO02 / 311140A1; PCTWO02 / 30954A1; Potillengen (TM) technology (Biowa, Inc.Princeton, N.J.); GlycoMAb (TM) glycosylation genetic engineering techniques (GLYCART biotechnology AG, Zurich, Switzerland) including the methods described are not limited thereto. See, for example, WO00061739; EA01229125; U.S. Patent Publication No. 200301115614; Okazaki et al, 2004, JMB, 336: 1239-49.

  It is also known in the art that glycosylation of the Fc region can be modified to increase or decrease effector function (eg, Umana et al, 1999, Nat. Biotechnol 17: 176-180; Davies et al, 2001, Biotechnol Bioeng 74: 288-294; Shields et al, 2002, J Biol Chem 277: 26733-26740; Shinkawa et al, 2003, J Biol Chem 278: 3466-3473, US Patent No. 6,602,684; No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCTWO 00/61739 A1, PCT WO 01/292246 A1, PCT WO 02 / 311140A PCTWO02 / 30954A1; Potillent ™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb ™ glycosylation genetic engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). Thus, in one embodiment, the Fc region of an antibody of the invention comprises an altered glycosylation of amino acid residues. In another embodiment, altered glycosylation of amino acid residues results in decreased effector function. In another embodiment, altered glycosylation of amino acid residues increases effector function. In a specific embodiment, the Fc region reduces fucosylation. In another embodiment, the Fc region is afucosylated (see, eg, US Patent Application Publication No. 2005/0226867).

2 shows a bar graph showing the effect of IgG1 DLL4 antibody on inhibition of HUVEC proliferation by DLL4 stimulation. Data are representative of independent experiments with n> 2. Figure 6 shows a bar graph showing the effect of IgG2 / 4 DLL4 antibody on in vitro HUVEC tube formation as assessed by measuring vessel length (mm) and # branching. 2 shows a bar graph showing the effect of an unlabeled anti-DLL4 antibody that displaces Alexa-647 labeled 21H3RK binding at a concentration of 0.1 μg / ml as measured by FACS analysis. A linear illustration of 12 chimeric DLL4 / DLL1 variants is shown. All variants encode the extracellular region of human DLL4 with human DLL1 substituting portions of individual subregions or binding regions as indicated. 2 shows a line graph showing 21H3RK binding to a chimeric knockout (“KO”) variant encoding DLL4 with a portion of the extracellular region replaced with the corresponding DLL1 region. A chimeric knockout ("KO") variant that encodes DLL4 having a portion of the extracellular region that is replaced with the corresponding DLL1 region, and a chimeric knockin ("KI") that encodes DLL1 having a region that is replaced with the DLL4 counterpart A line graph showing binding of 21H3RK to variants is shown.

  Embodiments of the invention relate to a novel set of DLL4 blocking molecules such as antibodies that inhibit Notch receptor signaling, for example. Such molecules can be used as single agents or in combination with other binding antibodies / binding agents. These molecules can be used in combination with any standard or novel anticancer agent.

  Embodiments of the invention relate to targeted binding agents that bind to DLL4. In some embodiments, the targeted binding agent binds to DLL4 and inhibits DLL4 binding to DLL4 Notch receptors (such as Notch 1, Notch 2, Notch 3, and / or Notch 4). In some embodiments, this binding may neutralize, block, inhibit, inactivate, or interfere with one or more aspects of DLL4-related effects. In one embodiment, the targeted binding agent is a monoclonal antibody, or a binding fragment thereof. Such monoclonal antibodies may be referred to herein as anti-DLL4 antibodies.

  Other embodiments of the invention include therapeutically effective fully human anti-DLL4 antibodies and antibody formulations. In one embodiment, the preparation of an anti-DLL4 antibody of the invention comprises a strong binding affinity for DLL4, the ability to block DLL4 receptor-ligand interactions, the ability to block DLL4-mediated signaling, endothelial cell proliferation and non-functional Ability to promote blood vessel formation, ability to regulate pericyte recruitment to blood vessels, ability to inhibit tumor growth, ability to increase tumor hypoxia / necrosis, alter endothelial fistula / stalk cell fate It has desirable therapeutic properties including ability and ability to modulate tumor cell survival or cancer stem cell survival and self-renewal.

  Furthermore, embodiments of the invention include methods of using the antibodies described herein to treat diseases. The anti-DLL4 antibodies of the present invention are useful for preventing DLL4-mediated tumor formation and tumor invasion into healthy tissue. Furthermore, DLL4 antibodies may be useful for treating diseases related to neoplastic diseases along with angiogenesis such as eye diseases such as AMD, inflammatory diseases such as rheumatoid arthritis, and cardiovascular diseases and sepsis. Any disease characterized by any type of malignancy, including metastatic cancer, lymphoma, and hematological cancer can also be treated by this inhibitory mechanism. Typical human cancers are bladder tumor, breast tumor, prostate tumor, basal cell cancer, biliary tract cancer, bladder cancer, osteosarcoma, brain and CNS cancer (eg glioma tumor), cervical cancer, choriocarcinoma, colon and Rectal cancer, connective tissue cancer, gastrointestinal cancer; endometrial cancer, esophageal cancer; eye cancer; head and neck cancer; gastric cancer; intraepithelial neoplasia; renal cancer; laryngeal cancer; leukemia; Non-small cell) lymphoma including lung cancer, Hodgkin and non-Hodgkin lymphoma; melanoma; myeloma, neuroblastoma, oral (eg, lips, tongue, mouth, and pharynx) cancer, ovarian cancer; pancreatic cancer, retinoblast Rhabdomyosarcoma; Rectal cancer, renal cancer, respiratory cancer; sarcoma, skin cancer; gastric cancer, testicular cancer, thyroid cancer; uterine cancer, urological cancer, and other cancers and sarcomas. Malignant diseases commonly diagnosed in dogs, cats, and other pets are lymphosarcoma, osteosarcoma, breast tumor, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, bronchial gland Cancer, fibroma, myxochondroma, lung sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing sarcoma, Wilms tumor, Burkitt lymphoma, microglioma, neuroblastoma, giant cell tumor of the bone, Oral tumor, fibrosarcoma, osteosarcoma and rhabdomyosarcoma, reproductive squamous cell carcinoma, infectious sexually transmitted disease tumor, testicular tumor, seminoma, Sertoli cell tumor, perivascular cell tumor, histiocytoma, melanoma ( E.g. granulocytic sarcoma), corneal papilloma, squamous cell carcinoma of the cornea, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal cancer, oral papilloma, hemangioendothelioma and cystadenoma, Follicular lymphoma Intestinal lymphosarcoma, including fibrosarcoma and pulmonary squamous cell carcinoma but not limited to. Typical rodent cancers such as ferrets include insulinoma, lymphoma, sarcoma, neuroma, islet cell tumor, gastric MALT lymphoma and gastric adenocarcinoma. Tumors affecting agricultural livestock are leukemia, periangiocytoma and bovine eye tumors in cattle; foreskin fibrosarcoma, ulcer squamous cell carcinoma, foreskin cancer, connective tissue tumor and mastocytoma in horses; liver in pigs Cell carcinoma; lymphoma and pulmonary adenomatosis in sheep; pulmonary sarcoma, lymphoma, rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphocytic leukemia in birds; retinoblastoma in liver Abnormal growth, lymphosarcoma (lymphoblastic lymphoma), plasma cell leukemia and buoyant sarcoma; dry buttery lymphadenitis (CLA) (chronic sheep and goat disease caused by bacterial corynebacterium pseudotuberculosis, infectious disease) , Infectious diseases) as well as sheep infectious lung tumors caused by Yargzikte.

  Other embodiments of the invention include diagnostic assays for specifically determining the amount of DLL4 in a biological sample. The assay kit can include the targeted binding agent or antibody disclosed herein along with the labels necessary to detect such antibodies. These diagnostic assays are useful for screening cell adhesion, cell invasion, angiogenesis, or proliferation related diseases including but not limited to neoplastic diseases.

  Another aspect of the invention is an antagonist of DLL4 biological activity, which antagonist binds to DLL4. In one embodiment, the antagonist is a targeted binding agent such as an antibody. The antagonist may be selected from the antibodies described herein, for example, antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

  In one embodiment, an antagonist of biological activity of DLL4 binds to DLL4, thereby inhibiting or suppressing DLL4 binding to Notch, resulting in cell adhesion and / or cell invasion and / or angiogenesis and / or Growth can be inhibited.

  One embodiment is a targeted binding agent that binds to the same epitope or epitopes as fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. is there.

  One embodiment is an antibody that binds to the same epitope or epitopes as fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK.

  One embodiment is a hybridoma that produces the targeted binding agent described herein above. One embodiment is a hybridoma that produces the light and / or heavy chains of the antibodies described herein above. In one embodiment, the hybridoma produces the light and / or heavy chain of a fully human monoclonal antibody. In another embodiment, the hybridoma produces light and / or heavy chains of fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. To do. Alternatively, the hybridoma produces an antibody that binds to the same epitope or epitopes as the fully human monoclonal antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK. Can do.

  Another embodiment is a nucleic acid molecule encoding a targeted binding agent as described herein above. In one embodiment, the nucleic acid molecule encodes the light or heavy chain of an antibody as described herein above. In one embodiment, the nucleic acid molecule encodes the light chain or heavy chain of a fully human monoclonal antibody. Yet another embodiment encodes the light chain or heavy chain of a fully human monoclonal antibody selected from antibodies 4B4, 2H10, 21F7, 12A1, 17F3, 9G8, 20G8, 21H3, 1E4, 3A7, 4B3, 1D4, or 21H3RK Nucleic acid molecule.

  Another embodiment of the invention is a vector comprising one or more nucleic acid molecules as described herein above, which vector encodes a targeted binding agent as defined above. In one embodiment of the invention, the vector comprises one or more nucleic acid molecules as defined herein above, which vector encodes the antibody light and / or heavy chain as defined above.

  Yet another embodiment of the invention is a host cell comprising a vector as described herein above. Alternatively, the host cell can contain more than one vector.

  Furthermore, one embodiment of the present invention produces a targeted binding agent of the present invention by culturing host cells under conditions in which the nucleic acid molecule is expressed to produce the targeted binding agent and subsequently recovers the targeted binding agent. It is a method to do. One embodiment of the invention is a method of producing an antibody of the invention by culturing a host cell under conditions in which a nucleic acid molecule is expressed to produce the antibody and then the antibody is recovered.

  In one embodiment, the present invention transfects at least one host cell with at least one nucleic acid molecule encoding a targeted binding agent as described herein above, and expresses the nucleic acid molecule in the host cell; A method of making the targeted binding agent by isolating the targeted binding agent. In one embodiment, the present invention transfects at least one host cell with at least one nucleic acid molecule encoding an antibody as described herein above, and expresses the nucleic acid molecule in the host cell. A method of producing an antibody by isolating.

  According to another aspect, the present invention includes a method of antagonizing DLL4 biological activity by administering an antagonist as described herein above. The method selects an animal in need of treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation and administers to the animal a therapeutically effective amount of an antagonist of the biological activity of DLL4 Can include.

  Another aspect of the invention includes a method of antagonizing the biological activity of DLL4 by administering a targeted binding agent as described herein above. The method selects an animal in need of treatment for disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation and provides a therapeutically effective amount of a targeted binding agent that antagonizes the biological activity of DLL4. Administering to the animal.

  Another aspect of the invention includes a method of antagonizing DLL4 biological activity by administering an antibody as described herein above. The method selects an animal in need of treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation, and provides a therapeutically effective amount of the antibody that antagonizes the biological activity of DLL4. Administration to an animal can be included.

  According to another aspect of the present invention, treatment of disease-related cell adhesion and / or invasion and / or angiogenesis and / or proliferation in an animal by administering a therapeutically effective amount of an antagonist of DLL4 biological activity. Provide a way to do it. The method selects an animal in need of treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or cell proliferation, and applies a therapeutically effective amount of an antagonist of the biological activity of DLL4 to the animal. Administration.

  According to another aspect of the invention, disease-related cell adhesion and / or invasion and / or angiogenesis and / or angiogenesis in an animal is administered by administering a therapeutically effective amount of a targeted binding agent that antagonizes the biological activity of DLL4. Alternatively, a method for treating proliferation is provided. The method selects an animal in need of treatment for disease-related cell adhesion and / or invasion and / or angiogenesis and / or proliferation and provides a therapeutically effective amount of a targeted binding agent that antagonizes the biological activity of DLL4, Administration to the animal. The targeted binding agent can be administered alone or in combination with additional antibodies or chemotherapeutic agents or radiation therapy.

  According to another aspect, disease-related cell adhesion and / or invasion and / or angiogenesis and / or proliferation in an animal is administered by administering a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. Provide a method. The method selects an animal in need of treatment for disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation, and provides the animal with a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. Administration. The antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drugs or radiation therapy.

  According to another aspect, there is provided a method of treating cancer in an animal by administering a therapeutically effective amount of an antagonist of DLL4 biological activity. The method can include selecting an animal in need of treatment for cancer and administering to the animal a therapeutically effective amount of an antagonist that antagonizes the biological activity of DLL4. The antagonist can be administered alone or in combination with additional antibodies or chemotherapeutic drugs or radiation therapy.

  According to another aspect, a method of treating cancer in an animal is provided by administering a therapeutically effective amount of a targeted binding agent that antagonizes the biological activity of DLL4. The method can include selecting an animal in need of cancer treatment and administering to the animal a therapeutically effective amount of a targeted binding agent that antagonizes the biological activity of DLL4. The targeted binding agent can be administered alone or in combination with additional antibodies or chemotherapeutic agents or radiation therapy.

  According to another aspect, a method of treating cancer in an animal is provided by administering a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. The method can include selecting an animal in need of cancer treatment and administering to the animal a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. The antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drugs or radiation therapy.

  According to another aspect, a method is provided for reducing or inhibiting tumor cell proliferation, adhesion, invasion and / or angiogenesis in an animal by administering a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. The method comprises selecting an animal in need of reduction, inhibition or inhibition of proliferation, cell adhesion, invasion and / or angiogenesis, and administering to the animal a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. obtain. The antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drugs or radiation therapy.

  According to another aspect, a method is provided for reducing tumor growth and / or metastasis in an animal by administering a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. The method can include selecting an animal in need of reduced tumor growth and / or metastasis and administering to the animal a therapeutically effective amount of an antibody that antagonizes the biological activity of DLL4. The antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drugs or radiation therapy.

  According to another aspect of the invention, the use of an antagonist of DLL4 bioactivity for the manufacture of a medicament for the treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation. provide. In one embodiment, an antagonist of DLL4 biological activity is a targeted binding agent of the invention. In one embodiment, the antagonist of DLL4 biological activity is an antibody of the invention.

  According to another aspect of the present invention, there is provided an antagonist of the biological activity of DLL4 for use as a medicament for the treatment of disease associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation. In one embodiment, an antagonist of DLL4 biological activity is a targeted binding agent of the invention. In one embodiment, the antagonist of DLL4 biological activity is an antibody of the invention.

  According to another aspect of the invention, a targeted binding agent that antagonizes the biological activity of DLL4 for the manufacture of a medicament for the treatment of disease-related cell adhesion and / or invasion and / or angiogenesis and / or proliferation Use of the antibody is provided.

  According to another aspect of the invention, a targeted binding agent or antibody that antagonizes the biological activity of DLL4 for use as a medicament for the treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation. provide.

  According to another aspect of the invention, a targeted binding agent that antagonizes the biological activity of DLL4 for the manufacture of a medicament for the treatment of disease-related cell adhesion and / or invasion and / or angiogenesis and / or proliferation Use of the antibody is provided.

  According to another aspect of the invention, there is provided an antibody that antagonizes DLL4 biological activity for use as a medicament for the treatment of disease-associated cell adhesion and / or invasion and / or angiogenesis and / or proliferation.

  According to another aspect of the present invention, there is provided the use of an antagonist of DLL4 bioactivity for the manufacture of a medicament for treating cancer in a mammal. In one embodiment, this antagonist of DLL4 biological activity is a targeted binding agent of the invention. In one embodiment, the antagonist of DLL4 biological activity is an antibody of the invention.

  According to another aspect of the invention, there is provided an antagonist of DLL4 bioactivity for use as a medicament for treating cancer in a mammal. In one embodiment, this antagonist of DLL4 biological activity is a targeted binding agent of the invention. In one embodiment, the antagonist of DLL4 biological activity is an antibody of the invention.

  According to another aspect of the invention, there is provided the use of a targeted binding agent that antagonizes the biological activity of DLL4 for the manufacture of a medicament for treating cancer in a mammal.

  According to another aspect of the present invention, there is provided a targeted binding agent that antagonizes DLL4 biological activity for use as a medicament for treating cancer in a mammal.

  According to another aspect of the present invention, there is provided the use of an antibody that antagonizes the biological activity of DLL4 for the manufacture of a medicament for treating cancer in a mammal.

  According to another aspect of the present invention, there is provided an antibody that antagonizes the biological activity of DLL4 for use as a medicament for treating cancer in a mammal.

  According to another aspect, the use of a targeted binding agent or antibody that antagonizes the biological activity of DLL4 for the manufacture of a medicament that reduces or inhibits proliferation, cell adhesion, invasion and / or angiogenesis in an animal. provide.

  According to another aspect, provided is a targeted binding agent or antibody that antagonizes the biological activity of DLL4 for use as an agent that reduces or inhibits proliferation, cell adhesion, invasion, and / or angiogenesis in an animal.

  According to another aspect, there is provided the use of a targeted binding agent or antibody that antagonizes the biological activity of DLL4 for the manufacture of a medicament that reduces tumor growth and / or metastasis in an animal.

  According to another aspect, provided is a targeted binding agent or antibody that antagonizes the biological activity of DLL4 for use as an agent that reduces tumor growth and / or metastasis in an animal.

  In one embodiment, the present invention is particularly suitable for antagonizing DLL4 in patients with tumors that are dependent or only dependent on DLL4.

  According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an antagonist of DLL4 bioactivity and a pharmaceutically acceptable carrier. In one embodiment, the antagonist comprises an antibody. According to another aspect of the present invention, there is provided a pharmaceutical composition comprising an antagonist of DLL4 bioactivity and a pharmaceutically acceptable carrier. In one embodiment, the antagonist comprises an antibody.

  In some embodiments, after administration of an antibody that specifically binds DLL4, a detergent is administered to remove excess circulating antibody from the blood.

  Anti-DLL4 antibodies are useful for the detection of DLL4 in patient samples and are therefore useful as a diagnosis of the medical conditions described herein above. Furthermore, based on the ability to significantly inhibit DLL4-mediated signaling activity (as demonstrated in the Examples below), anti-DLL4 antibodies have therapeutic effects in treating signs and symptoms resulting from DLL4 expression. Have. In specific embodiments, the antibodies and methods herein relate to the treatment of symptoms resulting from DLL4 induced cell adhesion, invasion, angiogenesis, proliferation and / or intracellular signaling. Further embodiments include melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocyte (liver) cancer, thyroid tumor, gastric (stomach) cancer, prostate cancer, breast cancer, ovarian cancer To treat cell adhesion, invasion, angiogenesis and / or proliferation related diseases including neoplastic diseases such as bladder cancer, lung cancer, glioblastoma, endometrial cancer, kidney cancer, colon cancer, and pancreatic cancer Using the antibodies and methods described herein above. The antibodies are also useful in diseases involving cell adhesion and / or invasion, atherosclerosis and angiogenesis in arthritis.

  In certain embodiments, the anti-DLL4 antibody or targeted binding agent is in a small population of stem cells with the ability to grow and efficiently generate additional tumor stem cells, such as acute myeloid leukemia (AML) and breast tumors. Can have therapeutic effects in the treatment of developing solid tumors.

  Another embodiment of the invention includes an assay kit that detects DLL4 in mammalian tissues, cells, or body fluids to screen for cell adhesion, invasion, angiogenesis, or proliferation-related diseases. The kit includes a targeted binding agent that binds to DLL4 and, if present, means for indicating the reaction of the targeted binding agent with DLL4. In one embodiment, the targeted binding agent that binds DLL4 is labeled. In another embodiment, the targeted binding agent is an unlabeled agent and the kit further comprises a means for detecting the targeted binding agent. Preferably, the targeted binding agent is labeled with a marker selected from the group consisting of fluorescent dyes, enzymes, radionuclides and radiopaque materials.

  Another embodiment of the invention includes an assay kit that detects DLL4 in mammalian tissues, cells, or body fluids to screen for cell adhesion, invasion, angiogenesis, or proliferation-related diseases. The kit includes an antibody that binds to DLL4 and, if present, means for indicating the reaction of the antibody with DLL4. The antibody can be a monoclonal antibody. In one embodiment, the antibody that binds DLL4 is labeled. In another embodiment, the antibody is an unlabeled primary antibody and the kit further comprises a means for detecting the primary antibody. In one embodiment, the means includes a labeled secondary antibody that is an anti-immunoglobulin. Preferably, the antibody is labeled with a marker selected from the group consisting of fluorescent dyes, enzymes, radionuclides and radiopaque materials.

  Additional embodiments, features, etc. relating to the antibodies disclosed herein are provided in the additional details below.

SEQUENCE LISTING Embodiments of the invention include specific antibodies listed in Table 1 below. The table shows the recognition number of each anti-DLL4 antibody along with the corresponding heavy and light chain genes and the variable region SEQ ID NO of each polypeptide. Each antibody was given a recognition number.

Table 2 is a table comparing the antibody heavy chain region to the heavy chain region of allogeneic germ cells and the kappa light chain region to the light chain region of allogeneic germ cells.

Definitions Unless otherwise defined, scientific and technical terms used herein have meanings as commonly understood by a person of ordinary skill in the art. Further, unless otherwise required by text, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature and techniques utilized in connection with cell and tissue culture, molecular biology, and protein chemistry, oligo or polynucleotide chemistry, and hybridization as described herein above are well known in the art. Are commonly used.

  Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's instructions or as commonly accomplished in the art or as described herein above. The foregoing techniques and procedures are generally described in accordance with conventional methods well known in the art and in various general and more specific references that are cited and explained throughout this specification. Went so. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001)). The nomenclature used in connection with analytical chemistry, organic synthetic chemistry, and medicinal chemistry described herein, and their testing methods and techniques are well known and commonly used in the art. is there. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, pharmaceutical formulation, and drug delivery, and treatment of patients.

  As utilized in accordance with the present disclosure, the following terms are understood to have the following meanings unless otherwise indicated.

  An antagonist or inhibitor can be a polypeptide, nucleic acid, carbohydrate, lipid, low molecular weight compound, oligonucleotide, oligopeptide, RNA interference (RNAi), antisense, recombinant protein, antibody, or a fragment or conjugate thereof or They can be their fusion proteins. For reviews of RNAi, see Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec: 55 (4): 629-48. Review) as a review of antisense by Opalinska JB, Gewirtz AM. (Sci STKE. 2003 Oct 28: 2003 (206): pe 47.).

  A compound refers to any low molecular weight compound having a molecular weight of less than about 2000 Daltons.

  The term “DLL4” refers to a molecule known as DLL4 protein, also delta-like protein 4 precursor, Drosophila delta homolog 4, hdelta 2, MGC126344, or UNQ1895 / PRO4341. The terms “neutralize” or “inhibit” when referring to a targeted binding agent, such as an antibody, relate to the ability of the antibody to eliminate, reduce or significantly reduce the activity of the target antigen. Thus, a “neutralizing” anti-DLL4 antibody of the present invention is capable of eliminating or significantly reducing DLL4 activity. A neutralizing DLL4 antibody can act, for example, by blocking the binding of native DLL4 to the DLL4 receptor Notch, eg, Notch-1 or Notch-4. By blocking this binding, the activity mediated by DLL4 signaling is significantly or completely eliminated. Ideally, neutralizing antibodies against DLL4 antagonism promote EC proliferation. Neutralizing DLL4 antibodies enhance angiogenesis, for example by promoting non-functional angiogenesis.

  An “antagonist of biological activity of DLL4” can eliminate, reduce or significantly reduce DLL4 activity. An “antagonist of biological activity of DLL4” can eliminate, reduce or significantly reduce DLL4 signaling. An “antagonist of biological activity of DLL4” can eliminate or significantly reduce angiogenesis and / or proliferation.

  “Decrease DLL4 signaling” means at least 5%, at least 10%, at least 15%, at least 20% compared to the amount of signaling in the absence of the targeted binding agent, antibody or antagonist of the present invention, At least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, At least 90%, at least 95% reduction in DLL4 signaling.

  An “optimized” sequence is an antibody sequence that has been mutated so that the non-germline sequence is mutated back to one or more residues relative to the germline sequence (for any of the antibodies described herein). Variable heavy chain or variable light chain) and may further include the removal of structural disturbances from sequences such as glycosylation sites or unpaired cysteines.

  The term “polypeptide” is used herein as a general term to denote a natural protein, fragment, or analog having a polypeptide sequence. Natural proteins, fragments, and analogs are therefore species of the polypeptide genus. Preferred polypeptides according to the present invention are light chain immunoglobulin molecules, such as kappa or lambda light chain immunoglobulin molecules, and combinations comprising fragments and analogs thereof and heavy chain immunoglobulin molecules, and vice versa. Human heavy chain immunoglobulin molecules and human kappa light chain immunoglobulin molecules, and antibody molecules. Preferred polypeptides according to the present invention may also simply comprise human heavy chain immunoglobulin molecules or fragments thereof.

  The term “natural” or “naturally occurring” as applied to an object and used herein refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified in the laboratory by humans or otherwise is naturally occurring.

  As used herein, the term “operably linked” refers to the location of a component that is represented in a relationship that allows the component to function in the intended manner of the component. For example, a control sequence “operably linked” to a coding sequence is combined in such a way that the coding sequence is expressed under conditions compatible with the control sequence.

  As expressed herein, the term “polynucleotide” refers to a polymeric form of nucleotides (either ribonucleotides or deoxyribonucleotides) at least 10 bases in length, or a modified form of any kind of nucleotide, or RNA -Means DNA heteroduplex. The term includes single and double stranded forms of DNA.

  As represented herein, the term “oligonucleotide” includes naturally occurring and modified nucleotides linked together by naturally occurring and non-naturally occurring bonds. Oligonucleotides are a polynucleotide subset generally comprising a base length of 200 bases or fewer. Preferably, the oligonucleotide is 10-60 bases in length, and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20-40 bases in length. Oligonucleotides are usually single stranded, for example in the case of probes, but oligonucleotides can be double stranded, for example when used in the construction of genetic variants. Oligonucleotides can be either sense or antisense oligonucleotides.

  As expressed herein, the term “naturally occurring nucleotides” includes deoxyribonucleotides and ribonucleotides. As expressed herein, the term “modified nucleotide” includes nucleotides having modified or substituted sugar groups and the like. As represented herein, the term “oligonucleotide linkage” refers to phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanithioate, Includes oligonucleotide linkages such as phosphoranidate, phosphoramidate. For example, LaPlanche et al. Nucl. Acids Res. 14: 9081 (1986); Stec et al. J. Am. Chem. Soc. 106: 6077 (1984); 1988); Zon et al. Anti-Cancer Drug Design 6: 539 (1991); Zon et al. )); Stec et al. US Pat. No. 5,151,510; Uhlmann and Peyman Chemical R See reviews 90: 543 (1990), the disclosures of which are incorporated herein by reference. If necessary, the oligonucleotide may include a label for detection.

  As represented herein, the term “selectively hybridize” means detectable and specific binding. Polynucleotides, oligonucleotides and fragments thereof selectively hybridize with nucleic acid strands under hybridization and wash conditions that minimize appreciable amounts of detectable binding to non-specific nucleic acids. High stringency conditions are known in the art and can be used to achieve selective hybridization conditions as described herein. In general, the nucleic acid sequence homology between the desired polynucleotide, oligonucleotide, or antibody fragment and the nucleic acid sequence is at least 80%, more generally preferably at least 85%, 90%, 95%, 99% , And 100% homology.

  Stringent hybridization conditions include hybridization to membrane bound DNA in approximately 45 ° C., 6 × sodium chloride / sodium citrate (SSC) (0.9 M sodium chloride, 90 mM sodium citrate, pH 7.0), Followed by one or more washes in about 50-65 ° C., 0.2 × SSC, 0.1% SDS, with higher stringency conditions in about 45 ° C., 6 × SSC. Hybridization to membrane bound DNA followed by one or more washes in about 60 ° C., 0.1 × SSC, 0.2% SDS, or any known in the art Other stringent hybridization conditions, including but not limited to Ausubel, FM et al, eds. 1989 urrent Protocols in Molecular Biology, vol.1, Green Publishing Associates, Inc.and John Wiley and Sons, Inc, reference NY p6.3.1~6.3.6 and 2.10.3). Two amino acid sequences are “homologous” if there is a partial or complete identity between the two amino acid sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. A gap (present in either of the two matched sequences) can maximize the match, preferably a gap length of 5 or less, more preferably a gap length of 2 or less. Alternatively and preferably, if two protein sequences have an alignment score greater than 5 (in standard deviation units) using the ALIGN program with a mutation data matrix and a gap penalty of 6 or more, the two protein sequences ( Or polypeptide sequences derived from them that are at least about 30 amino acids in length) are homologous when this term is used herein. Dayhoff, M.O, in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5, National Biomedical Research Foundation (1972) and Supplement 10 to this volume). Two sequences or portions thereof are more preferably homologous when these amino acids are 50% or more identical when optimally aligned using the ALIGN program. It should be understood that there can be different regions of homology between two orthologous sequences. For example, the functional sites of mouse and human orthologs can have higher homology than non-functional sites.

  As used herein, the term “corresponds to” the polynucleotide sequence is homologous to all or part of the reference polynucleotide sequence (ie, is identical and not strictly evolutionary related). Or that the polypeptide sequence is identical to the reference polypeptide sequence.

  In contrast, as used herein, the term “complementary to” means that the complementary sequence is homologous to all or part of a reference polynucleotide sequence. As an example, the nucleotide sequence “TATAC” corresponds to the reference sequence “TATAC” and is complementary to the reference sequence “GTATA”.

  The term “sequence identity” means that two polynucleotide or amino acid sequences are identical (ie, on a nucleotide-by-nucleotide or residue-by-residue basis) on a comparison frame. The term “percent sequence identity” is used to compare two sequences that are optimally aligned on a comparison frame and to obtain identical number of positions (eg, A, T, C, G) , U, or I) or the number of positions where amino acid residues occur in both sequences, and the number of matched positions divided by the total number of positions in the comparison frame (ie, frame size), and the percentage of sequence identity Is calculated by multiplying the result by 100 to obtain As used herein, the term “substantial identity” refers to a property of a polynucleotide or amino acid sequence, as compared to a reference sequence on a comparison frame at a position of at least 18 nucleotides (6 amino acids): Frequently, polynucleotides and amino acids are at least 85% sequence identity, preferably at least 90-95% sequence identity, compared to a reference sequence on the frame of at least 24-48 nucleotides (8-16 amino acids) position A percentage of sequence identity comparing the reference sequence to a sequence that may comprise a total of 20% or less of deletions or additions of the reference sequence on the comparison frame. Is calculated by The reference sequence can be a subset of a longer sequence.

As used herein, the twenty standard amino acids and their abbreviations follow standard usage. ^{Immunology-A Synthesis (2 nd Edition} , E.S.Golub and D.R.Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)) reference is made to the detailed, which is incorporated herein by reference . Twenty standard amino acid stereoisomers (eg, D-amino acids), non-natural amino acids such as α-, α-2 substituted amino acids, N-alkyl amino acids, lactic acid, and other non-standard amino acids are: It can also be a suitable component of a polypeptide of the invention. Examples of non-standard amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

  Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5 'end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The addition of the nascent RNA transcript in the 5 ′ to 3 ′ direction is referred to as the transcription direction. The sequence region on the DNA strand having the same sequence as RNA and the sequence region on the 3 ′ end side of the RNA transcript are referred to as “downstream sequence”.

  As applied to a polypeptide, the term “substantially identical” means that two peptide sequences, when optimally aligned, for example, by a program GAP or BESTFIT using a gap weight initial value, Means sharing at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, most preferably at least 99% sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions. Conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, the group of amino acids having an aliphatic side chain is glycine, alanine, valine, leucine, and isoleucine, and the group of amino acids having an aliphatic hydroxyl side chain is serine and threonine, having an amide-containing side chain The group of amino acids is asparagine and glutamine, the group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan, and the group of amino acids having basic side chains is lysine, arginine, and histidine A group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

  As explained herein, minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are intended to be included in the present invention, and changes in the amino acid sequence may be associated with an antibody or immunity described herein. Maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% sequence identity to the globulin molecule. In particular, conservative amino acid substitutions are contemplated. A conservative substitution is a substitution that replaces within a family of amino acids having related side chains. Genetically encoded amino acids are generally (1) acidic = aspartic acid, glutamic acid; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine. , Methionine, tryptophan; and (4) uncharged polarity = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. More preferred families: serine and threonine are aliphatic hydroxy families, asparagine and glutamine are amide-containing families, alanine, valine, leucine and isoleucine are aliphatic families, and phenylalanine, tryptophan, and tyrosine are aromatic It is a family. For example, leucine isoleucine or valine, aspartic glutamic acid, threonine serine, isolated substitution, or similar substitution with amino acids structurally related to amino acids, if the substitution is a frame site It is reasonable to expect that if the amino acids are not involved, they do not have a serious effect on the binding function or properties of the resulting molecule. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. The assay is described in detail herein. Those skilled in the art can readily prepare fragments or analogs of antibodies or immunoglobulin molecules. The preferred amino and carboxy termini of multiple fragments or analogs are near the boundaries of the functional region. Structural and functional regions can be identified by comparing nucleotide and / or amino acid sequence data to public or private sequence databases. Preferably, computer-controlled comparison methods are used to identify sequence motifs or predicted protein conformation regions that are present in other proteins with known structure and / or function. Methods for identifying protein sequences that are folded into a known three-dimensional structure are known. Bowie et al. Science 253: 164 (1991). Thus, the foregoing examples show that one skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional regions according to the antibodies described herein.

  Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. Deamidated forms of these residues are included within the scope of the present invention.

  In general, cysteine residues in proteins are either involved in cysteine-cysteine disulfide bonds or are sterically protected from disulfide bond formation when they are part of a folded protein region. Protein disulfide bond formation is a complex process determined by environmental redox potentials and specialized enzymes in thiol disulfide exchange (Creighton, Methods Enzymol. 107,305-329, 1984; Houee-Levin, Methods Enzymol. 353). 35-44, 2002). When a cysteine residue is unpaired in a protein structure and is not sterically protected by folding, it forms a disulfide bond with a free cysteine in solution in a process known as disulfide shuffling. be able to. In another process known as disulfide scrambling, free cysteines also interfere with naturally occurring disulfide bonds (eg, disulfide bonds present in antibody structures), weak bonds, and low biological activity. And / or exhibit low stability.

  Preferred amino acid substitutions are (1) reduce vulnerability to proteolysis (2) reduce vulnerability to oxidation (3) change binding affinity of protein complex formation (4) change binding affinity, and ( 4) A substitution that imparts or modifies other physicochemical or functional properties of the analog. Analogs can include various mutations in sequences other than the naturally occurring peptide sequences. For example, one or multiple amino acid substitutions (preferably conservative amino acid substitutions) can occur within a naturally occurring sequence (preferably in the portion of the polypeptide outside the region that forms the intermolecular contact). Conservative amino acid substitutions should not substantially change the structural characteristics of the parent sequence (e.g., replacement amino acids cleave the helix that occurs in the parent sequence, or other types of secondary structures that characterize the parent sequence). Should not tend to destroy). Examples of secondary and tertiary structures of polypeptides approved in the art are Proteins, Structures and Molecular Principles (Creighton, Ed, W. H. Freeman and Company, New York (1984)); Introductor (C. Branden and J. Tooze, eds, Garland Publishing, New York, NY (1991)); and Thornton et a. Nature 354: 105 (1991), each of which is incorporated herein by reference. Incorporated into.

  In addition, such methods include substitution or deletion of amino acids at one or more cysteine residues in the variable region that are involved in intrachain disulfide bonds to produce antibody molecules that lack one or more disulfide bonds within the chain. Can be used to produce.

  The term “CDR region” or “CDR” is intended to indicate the hypervariable regions of the heavy and light chains of an antibody that confer antigen binding specificity to the antibody. CDR is Kabat System (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Human Edition, SevereH, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services, Human Services obtain. An antibody generally comprises three heavy chain CDRs and three light chain CDRs. In this case, the term CDR or CDRs refers to one of these regions or some of these regions containing most of the amino acid residues involved in the antibody's affinity binding to the antigen or epitope recognized by the antibody or Used herein to show the whole.

  The third CDR of the heavy chain (HCDR3) has greater size variability (basically greater diversity due to the mechanism of the gene sequence that causes it). The longest known size is 26, but HCDR3 can be as short as two amino acids. CDR length can also vary, especially with the length that can be accommodated by the basic framework. Functionally, HCDR3 plays a partly important role in determining antibody specificity (Segal et al., PNAS, 71: 4298-4302, 1974, Amit et al., Science, 233: 747). -753, 1986, Chothia et al, J. Mol. Biol., 196: 901-917, 1987, Chothia et al., Nature, 342: 877-883, 1989, Caton et al., J. Immunol., 144. 1965-1968, 1990, Sharon et al., PNAS, 87: 4814-4817, 1990, Sharon et al., J. Immunol., 144: 4863-4869, 1990, Kabat et al, J. Immunol., 147 : 1709-1719, 1991).

  As expressed herein, the term “CDR set” includes CDR1, CDR2 and CDR3. Thus, the HCDR set represents HCDR1, HCDR2 and HCDR3, and the LCDR set represents LCDR1, LCDR2 and LCDR3.

  VH and VL regions of the invention and variants of CDRs, including those variants whose amino acid sequences are described herein and available for targeting agents and antibodies to DLL4, can be obtained by methods of sequence variation or sequence variation and as desired. It can be obtained by methods of screening for antigens to target using features. Examples of desirable characteristics include increased binding affinity of the antigen for known antibodies specific for the antigen and, if known, increased neutralization of antigen activity for known antibodies specific for the antigen, specific The specific competitive ability of a known antibody or ligand to an antigen at a specific molar ratio, the ability to immunoprecipitate a ligand-receptor complex, the ability to bind to a specified epitope, a linear epitope, For example, peptide binding scans, eg, peptide sequences identified using screened peptides in linear and / or restricted conformations, formed by discrete residues Ability to bind conformational epitopes, ability to modulate new biological activity of DLL4 or downstream molecules, binds to DLL4 And / or the ability to neutralize DLL4, and / or any other desired property.

  Techniques required to make substitutions in the amino acid sequences of CDRs, antibody VH or VL regions and antigen binding sites are available in the art. Variants of the antibody molecules disclosed herein can be produced and used in the present invention. A precedent of computational chemistry by applying multivariate data analysis techniques to structure / property-activity relationship (Wold, et al. Multivariate data analysis and Chemistry. Chemistry. Chemistry. Chemistry. Chemistry. Chemistry. Chemistry. B. Kowalski), D. Reidel Publishing Company, Dordrecht, Holland, 1984), followed by quantitative activity-property relationships of antibodies, patterns of classification and patterns of statistical regression, pattern recognition well known Derived using mathematical techniques (Norman et al. Applied Regression Analysis. Wiley-Interscience: 3rd edition (April 1998); May 11, 1995); Krzanowski, Wojtek.Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Sciences. cember 2000); Witten, Ian H. & Frank, Eibe Data Mining:.. Practical Machine Learning Tools and Techniques with Java Implementations Morgan Kaufmann; (October 11,1999): Denison David G. T. (Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith. Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Proficiency and Stability and Stability). ly 2002); Gose, Arup K. & Viswanadhan, Vellakad N. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications. In some cases, antibody properties can be derived from experience and theoretical models of antibody sequences (eg, analysis of promising contact residues or calculated physicochemical properties), functional and three-dimensional structures and their Properties can be considered alone and in combination.

  An antibody antigen binding site composed of a VH region and a VL region is generally formed by six loops of a polypeptide (three from the light chain variable region (VL) and three from the heavy chain variable region (VH)). Is done. Antibody analysis of known atomic structures has revealed the relationship between antibody binding site sequence and three-dimensional structure. These relationships suggest that, except for the third region (loop) of the VH region, the binding site loop has one of the canonical structures that are a few main chain conformations. It has been shown that the canonical structure formed within a particular loop is determined by its size and the presence of specific residues at key sites in both the loop and framework regions.

  This study of sequence-structure relationships is important for maintaining the three-dimensional structure of the CDR loop of an antibody, and thus the residues of a known sequence but with an unknown three-dimensional structure that maintain binding specificity. Can be used for prediction. These predictions can be supported by a comparison of the predictions against the results obtained from the main optimization experiments. In a structural approach, a model of the antibody molecule can be created at will either freely or using a commercial package such as WAM. Protein visualization and analysis software packages such as Insight II (Accelrys, Inc.) or Deep View can then be used to assess possible substitutions at each position of the CDR. This information can then be used to make substitutions that appear to have minimal or beneficial effects on activity or that would confer other desired properties.

  As used herein, the term “polypeptide fragment” has an amino-terminal and / or carboxy-terminal deletion, but the remaining amino acid sequence is naturally occurring, eg deduced from the full-length cDNA sequence. Represents a polypeptide that is identical to the corresponding position in the sequence. Generally, a fragment is at least 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acids long, more preferably at least 20 amino acids long, usually at least 50 amino acids And even more preferably a length of at least 70 amino acids. As used herein, the term “analog” has substantial identity with some deduced amino acid sequences and has at least one of the following properties: (1) DLL4 under appropriate binding conditions Represents a polypeptide comprising a portion of at least 25 amino acids having specific binding to (2) the ability to block proper VEGF / DLL4 binding or (3) the ability to inhibit DLL4 receptor tyrosine kinase activity. In general, polypeptide analogs contain conservative amino acid substitutions (or additions or deletions) with respect to the naturally occurring sequence. In general, an analog is at least 20 amino acids long, preferably at least 50 amino acids long, and often can be as long as a full-length naturally occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs that have properties similar to analogs of template peptides. These types of non-peptide compounds are referred to as “peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference). Such compounds have often evolved in favor of computer-controlled molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. In general, a peptidomimetic is structurally similar to an exemplary polypeptide such as a human antibody (ie, a polypeptide having biochemical or pharmacological properties), but —CH ₂ NH—, —CH _{2. S -, - CH 2 -CH 2} -, - CH = CH- ( cis and _{trans), - COCH 2 -, -} CH (OH) CH 2 -, and bond selected from the group consisting of _-CH 2 SO- Have one or more peptide bonds optionally substituted by methods well known in the art. Systematic substitution of one or more amino acids of the consensus sequence with the same type of D-amino acid (eg, D-lysine instead of L-lysine) can be used to produce more stable peptides. In addition, restricted peptides containing consensus sequences or variations of substantially identical consensus sequences may be obtained by methods known in the art (Rizzo and Giersch Ann. Rev. Biochem. 61: 387 (1992), hereby incorporated by reference. Can be produced, for example, by adding an internal cysteine residue that can form an intramolecular disulfide bridge that cyclizes the peptide.

  The antibodies are oligoclonal, polyclonal antibody, monoclonal antibody, chimeric antibody, CDR-conjugated antibody, multispecific antibody, bispecific antibody, catalytic antibody, chimeric antibody, humanized antibody, fully human antibody, anti-idiotype antibody, As well as antibodies that can be labeled in soluble or bound form, and fragments, variants or derivatives of antibodies alone or in combination with other amino acid sequences provided by well-known techniques. The antibody may be derived from any species.

  As used herein, the terms “antibody” and “antibodies” (immunoglobulin) refer to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, camelid antibodies (camelised antibodies). ) And chimeric antibodies. As used herein, the term “antibody” or “antibody” refers to a three-dimensional binding space having an internal surface shape and charge distribution that is complementary to the properties of the antigenic determinants of the antigen. Represents a polypeptide or group of polypeptides comprising at least one binding region formed from the folding of a polypeptide chain. Natural antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by a number of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end, the light chain constant region is aligned with the first constant region of the heavy chain, and the light chain variable region is the heavy chain variable region Align with. Light chains are classified as either lambda chains or kappa chains based on the amino acid sequence of the light chain constant region. The kappa light chain variable region may also be referred to herein as VK. The term “variable region” can also be used to describe a heavy or light chain variable region. Certain amino acid residues are thought to form an interface between the light and heavy chain variable regions. The variable regions of each light / heavy chain pair form the antibody binding site. Such antibodies can be generated from any mammal, including but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, and the like.

  The term “antibody” or “antibodies” includes binding fragments of the antibodies of the invention, exemplary fragments being single chain Fvs (scFv), single chain antibodies, single region antibodies. , Region antibody, Fv fragment, Fab fragment, F (ab ′) fragment, F (ab ′) 2 fragment, antibody fragment exhibiting a desired biological activity, disulfide fixed variable region (dsFv), dimer variable region (double Specific antibodies), from anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention), intracellular antibodies, linear antibodies, any of the above antibody fragments and epitope binding fragments Includes single chain antibody molecules and multispecific antibodies formed. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an antigen binding site. The immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Cleavage of the antibody by the enzyme papain results in two identical antigen-binding fragments, also known as “Fab” and “Fc” fragments, which have no antigen-binding activity but have the ability to crystallize. Cleavage of the antibody with the enzyme pepsin results in an “F (ab ′) ₂ ” fragment containing two antigen binding sites, with the two arms of the antibody molecule remaining linked. F (ab ′) ₂ fragments have the ability to crosslink antigens.

  “Fv” as used herein refers to the smallest fragment of an antibody that retains both antigen recognition and antigen binding sites. This region consists of a dimer of one heavy chain and one light chain variable region in a tight non-covalent or covalent association. In this arrangement, the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer. A total of 6 CDRs confer antigen binding specificity to the antibody. However, even a single variable region (or half of an Fv containing only three CDRs specific for the antigen) has a lower affinity than the entire binding site, but has the ability to recognize and bind to the antigen.

  “Fab” as used herein refers to a fragment of an antibody that comprises the constant region of the light chain and the CH1 region of the heavy chain.

  “DAb” as used herein refers to a fragment of an antibody that is the smallest functional binding unit of a human antibody. A “dAb” is a single region antibody, comprising either the heavy chain variable region (VH region) of an antibody or the light chain variable region (VL region) of an antibody. Each dAb contains three of the six naturally-occurring CDRs (Ward et al, Binding activities of a repertoire of single immunity bulco, et al., 1987). , Domain antibodies: protein for therapy, Trends Biotechnol. 21, 484-49 (2003)). They have a molecular weight in the range of 11-15 kDa, 4 times smaller than the antigen-binding (Fab) 2 fragment, and half the size of a single chain Fv (scFv) molecule.

  “Camelization”, as used herein, refers to an antibody molecule that consists of a heavy chain dimer that lacks a light chain but nevertheless has a broad antigen-binding repertoire (Hamers- Castellman C, Atarhouuch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendhamman N, Hamers R (1993) Naturally occupying anti-bodies4.

The term "bispecific antibody" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a light chain variable region in the same polypeptide chain _{(V H -V L) (V} L) Contains a heavy chain variable region (V _H ) that binds to By using a linker that is too short to pair between two regions on the same chain, those regions pair with the complementary region of another chain to create two antigen binding sites Will be forced. Bispecific antibodies are described in more detail, for example, in European Patent No. 404,097, WO 93/11161, and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). ing.

  It has been shown that intact antibody fragments can perform the function of binding antigens. Examples of binding fragments include (Ward, ES et al., (1989) Nature 341, 544-546) Fab fragments consisting of VL, VH, CL and CH1 regions, (McCafferty et al (1990) Nature, 348). , 552-554) Fb fragment consisting of VH and CH1 regions, (Holt et al (2003) Trends in Biotechnology 21,484-490) Fv fragment consisting of VL and VH regions of a single antibody, (iv) VH or VL region DAb fragments (Ward, ES, et al, Nature 341, 544-546 (1989), McCafferty et al (1990) Nature, 348, 552-554, Holt et al (2003) Trends in Biotechnologue). y 21,484-490), (v) an isolated CDR region, (vi) a bivalent fragment comprising two linked Fab fragments, (vii) a VH region and a VL region associate the two regions and bind antigen Single chain Fv molecules (scFv) linked by peptide linkers that allow site formation (Bird et al, (1988) Science, 242, 423-426, Huston et al, (1988) PNAS USA, 85, 5879 -5883), (viii) bispecific single chain Fv dimer (PCT / US92 / 09965) and (ix) "bispecific antibody", multivalent or multispecific fragments constructed by gene fusion (WO 94/13804, Holliger, P. (1993) et al, Proc. Natl. Acad. Sci. USA 90 6444-6. 48), and the like. Fv, scFv or bispecific antibody molecules can be stabilized by the incorporation of disulfide bridges linking the VH and VL regions (Reiter, Y. et al, Nature Biotech, 14, 1239-1245, 1996). Small antibodies containing scFv bound to the CH3 region can also be generated (Hu, S. et al, (1996) Cancer Res, 56, 3055-3061). Other examples of binding fragments include Fab ′ (which contains one or more cysteines from the antibody hinge region), which differs from the Fab fragment by the addition of several residues at the carboxy terminus of the heavy chain CH1 region, and the constant Fab′-SH, which is a Fab ′ fragment in which a cysteine residue in the region has a free thiol group, can be mentioned.

  The term “variable” refers to the fact that a particular portion of the variable region varies greatly in sequence between antibodies and is responsible for the binding specificity of each particular antibody for that particular antigen. However, variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in a section called complementarity determining regions (CDRs) of both the light chain and heavy chain variable regions. The more highly conserved part of the variable region is called the framework region (FR). The variable region of each of the natural heavy and light chains contains four FR regions, most of which have a β-sheet configuration, a loop connecting the β-sheet structure, and in some cases, the β-sheet structure It is linked by three CDRs that form a loop that forms part. The CDRs of each chain are grouped in close proximity by the FR region and with the CDRs of the other chain and contribute to the formation of the antigen binding site of the antibody (Kabat et al, Sequences of Proteins Interests, 5th Ed. Public Health , National Institutes of Health, Bethesda, MD (1991)). The constant region is generally not directly involved in antigen binding, but may affect antigen binding affinity and exhibit various effector functions such as antibody involvement in ADCC, CDC, and / or apoptosis.

  As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are associated with binding to an antigen. Hypervariable regions are amino acid residues of a “complementarity determining region” or “CDR” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable region) and Heavy chain variable region residues 31-35 (H1), 50-65 (H2) and 95-102 (H3); Kabat et al, Sequences of Proteins of Immunological Institute, 5th Ed. Public Health Institute, Nation Health Service, , Bethesda, MD (1991)) and / or those residues of the “hypervariable loop” (eg, residues 26-32 (Ll), 50-52 (L2) and 91-96 (L3) of the light chain variable region) ) And 26-32 (H1) of the heavy chain variable region 901-917 (1987)):; 53~55 (H2) and 96~101 (H3) Chothia and Lesk, J.Mol.Biol, 196. “Framework” or “FR” residues are those variable region residues flanking the CDRs. FR residues are found in chimeric antibodies, humanized antibodies, human antibodies, region antibodies, bispecific antibodies (diabodies), vaccine antibodies (linear antibodies), linear antibodies, and bispecific antibodies (bispecific antibodies). Exists.

  As used herein, terms such as a target binding agent, a target binding protein, a specific binding protein, etc. refer to an antibody or binding fragment thereof that selectively binds to a target site. In one embodiment, the targeted binding agent is specific for a single target site. In other embodiments, the targeted binding agent is specific for more than one target site. In one embodiment, the targeted binding agent is a monoclonal antibody and the target site can be an epitope.

“Binding fragments” of an antibody are produced by recombinant DNA techniques or by enzymatic or chemical degradation of intact antibodies. Binding fragments include Fab, Fab ′, F (ab ′) ₂ , Fv, dAb and single chain antibodies. Antibodies other than “bispecific” or “bifunctional” antibodies are understood to have the same respective binding site. The antibody substantially inhibits adhesion of the receptor to the counter-receptor, and excess antibody (as measured in an in vitro competitive binding assay) is at least about 20%, 40%, 60% or 80%, And greater than 80%, usually greater than about 85%, reduces the amount of receptor bound to the counterreceptor.

  The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitope determinants usually consist of chemically active surface populations of molecules such as amino acids or sugar side chains and may, but need not always, have specific three-dimensional structural characteristics and specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is 1 μM or less, preferably 100 nM or less and most preferably 10 nM or less.

  The term “drug” is used herein to indicate a chemical compound, a mixture of chemical compounds, a biopolymer, or an extract made from a biological material.

  “Active” or “activity” with respect to a DLL4 polypeptide refers to a portion of a DLL4 polypeptide that has the biological or immunological activity of the native DLL4 polypeptide. As used herein, “biological” refers to a biological function that results from the activity of a native DLL4 polypeptide. Preferred DLL4 biological activity includes, for example, DLL4 induced cell adhesion and cell invasion, and / or angiogenesis, and / or proliferation.

  “Mammal” when used herein refers to any animal that is considered a mammal. Preferably the mammal is a human.

  “Animal” as used herein includes animals that are considered mammals. Preferably the animal is a human.

  The term “mAb” refers to monoclonal antibody.

  “Liposome” as used herein refers to a vesicle that may be useful for delivery to a mammal of an agent that may comprise a DLL4 polypeptide of the invention or an antibody against such DLL4 polypeptide.

As used herein, “label” or “labeled” refers to a detectable moiety to a polypeptide, eg, a radiolabel, fluorescent label, enzyme label, chemiluminescent group or Represents the addition of a biotinyl group. The radioisotope or radionuclide can include ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, and the fluorescent label can include rhodamine, lanthanide phosphors or FITC The enzyme label may include horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase.

  Additional labels include, by way of example, glucose-6-phosphate dehydrogenase (“G6PDH”), α-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase. And enzymes such as peroxidases; dyes; additional fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorescent dyes, GFP (GFP of “green photoprotein”), dansyl, umbelliferone, phycoerythrin, phycocyanin , Allophycocyanin, o-phthalaldehyde, and fluorophores and chelates such as fluorescamine, lanthanide cryptate, such as europium (Perkin Elmer and (Cis Biointernational); chemiluminescent labels or chemiluminescence such as isoluminol, luminol and dioxetane; photosensitizer; coenzyme; enzyme substrate; particles such as latex or carbon particles; metal sol; crystal; liposome; dye; Or cells that can be further labeled with other detectable groups; molecules such as biotin, digoxigenin or 5-bromodeoxyuridine; for example, Pseudomonas aeruginosa exotoxin (PE or cytotoxic fragments or variants thereof), Diphtheria toxin or cytotoxic fragments or variants thereof, botulinum toxin A, B, C, D, E or F, ricin or cytotoxic fragments thereof, such as ricin A, abrin or cytotoxic fragments thereof, saporin or cytotoxicity thereof Fragment, pokeweed antiviral toxin or Cytotoxic fragments, and bryodin (bryodin) including one or toxin moiety such as a toxin moiety selected from the group of the cytotoxic fragment is not limited to them.

  As used herein, the term “pharmaceutical or pharmaceutical” refers to a chemical compound or chemical composition that can induce a desired therapeutic effect when properly administered to a patient. Other chemical terms herein are used according to conventional usage in the art, and include The McGraw-Hill Dictionary of Chemical Terms (Parker, S, Ed, McGraw-Hill, San Francisco (1985)) as an example. (Incorporated herein by reference).

  As used herein, “substantially pure” means that the subject species is present as the predominant species (ie, on a molar basis, more abundant in the composition than any other individual species. Preferably, the substantially purified fraction is a composition comprising at least about 50% of all macromolecular species in which the subject species is present (on a molar basis). In general, a substantially pure composition comprises more than about 80%, more preferably more than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. Most preferably, the species of interest is purified to a basic identity where the composition consists essentially of a single macromolecular species (contaminant species can be detected in the composition by conventional detection methods). Can not).

  The term “patient” includes human and veterinary subjects.

  “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to non-specific cytotoxic cells that express the IgFc receptor (FcR), such as natural killer (NK) cells, monocytes, neutrophils, and macrophages ) Represents a cell-mediated reaction that recognizes the bound antibody on the target cell and subsequently causes lysis of the target cell. Primary cells that mediate ADCC, NK cells express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay as described in US Pat. Nos. 5,500,362 or 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively and additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1988). “Complement dependent cytotoxicity” and “CDC” refer to the mechanism by which an antibody performs its cell death function. It is initiated by the binding of C1q, a component of the first component of complement, to the Fc region of Ig, IgG or IgM in complex with the antigen (Hughs-Jones, NC, and B Gardner. 1979. MoI. Immunol. 16: 697). C1q is a glycoprotein with a large complex structure of about 410 kDa that is present in human serum at a concentration of 70 μg / ml (Cooper, N.R. 1985. Adv. Immunol. 37: 151). Together with the two serine proteases C1r and C1s, C1q forms a complex C1, which is the first component of complement. At least two N-terminal globular heads of C1q must bind to Ig Fc for C1 activation and thus for initiation of the complement cascade (Cooper, N.R. 1985. Adv. Immunol. 37: 151).

  As used herein, the term “antibody half-life” refers to the pharmacokinetic properties of an antibody, which is a measure of the average survival time of an antibody molecule after antibody administration. Antibody half-life is, for example, 50% of the known amount of immunoglobulin from the patient's body or its specific compartment as measured in serum or plasma, ie circulating half-life, or in other tissues. It can be expressed as the time required to eliminate. Half-life may vary depending on the immunoglobulin or immunoglobulin class. In general, an increase in antibody half-life results in an increase in the mean residence time (MRT) in the circulation of the administered antibody.

  The term “isotype” refers to the classification of an antibody heavy or light chain constant region. The constant region of an antibody does not participate in antigen binding but exhibits various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further classified into subclasses (isotypes), eg, IgG1 (γ1), IgG2 (γ2), IgG3 (γ3), and IgG4 (γ4), and IgA1 and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The structures and three-dimensional configurations of different classes of immunoglobulins are well known. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3, IgG4 and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate in humans. Human light chain constant regions can be divided into two major classes, kappa and lambda.

  If desired, the isotype of an antibody that specifically binds DLL4 can be switched, for example, to take advantage of the biological properties of the different isotypes. For example, in some situations, in connection with the production of antibodies as therapeutic antibodies to DLL4, it is desirable that the antibodies can bind to complement and be involved in complement dependent cytotoxicity (CDC). obtain. There are several antibody isotypes that can do this, including but not limited to the following mouse IgM, mouse IgG2a, mouse IgG2b, mouse IgG3, human IgM, human IgA, human IgG1, and human IgG3. In other embodiments, in connection with the production of antibodies as therapeutic antibodies to DLL4, it is desirable that the antibodies can bind to Fc receptors on effector cells and be involved in antibody-dependent cellular cytotoxicity (ADCC). It can be. There are several isotypes of antibodies that can do this, including but not limited to the following mouse IgG2a, mouse IgG2b, mouse IgG3, human IgG1, and human IgG3. The antibody produced need not initially possess such an isotype; instead, the antibody produced can possess any isotype, after which this antibody is conventionally well known in the art. It will be understood that isotypes can be switched using this technique. Such techniques include, among others, recombinant techniques (see, eg, US Pat. No. 4,816,397), cell fusion techniques (eg, US Pat. Nos. 5,916,771 and 6,207,418). Including direct use).

  By way of example, the anti-DLL4 antibody discussed herein is a fully human antibody. If the antibody retains the desired binding to DLL4, it can be quickly switched isotype to produce human IgM, human IgG1, or human IgG3 isotypes (both antibody specificity and antibody affinity). Still have the same variable region (defining part). Such molecules can then bind to complement and participate in CDC and / or bind to Fc receptors on effector cells and participate in ADCC.

  A “whole blood assay” uses unfractionated blood as a source of natural effectors. Blood contains complement in plasma along with effectors of cells expressing FcR, such as polymorphonuclear cells (PMN) and mononuclear cells (MNC). Thus, the whole blood assay can simultaneously evaluate the synergistic effects of both in vitro ADCC and CDC effector mechanisms.

  As used herein, a “therapeutically effective” amount is an amount that provides some improvement or benefit to the subject. An otherwise defined “therapeutically effective” amount is an amount that provides some relief, alleviation, and / or reduction in at least one clinical symptom. Clinical symptoms associated with diseases that can be treated by the methods of the present invention are well known to those skilled in the art. Furthermore, those skilled in the art will appreciate that the therapeutic effect need not be a complete or curative effect as long as some benefit is provided to the subject.

  As used herein, the term “and / or” should be construed as a specific disclosure of each of the two specified features or components, whether or not there are others. is there. For example, “A and / or B” is a specific example of each of (i) A, (ii) B, and (iii) A and B, as if each were individually described herein. Should be construed as disclosure.

Antibody structure Basic antibody structural units are known to contain tetramers. Each tetramer consists of two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). . The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is primarily involved in antibody recognition. The carboxy terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, and the heavy chain further includes a “D” region of about 10 amino acids. See generally Fundamental Immunology Ch. 7 (Paul, W, ed., 2nd ed. Raven Press, NY (1989)), which is incorporated by reference in its entirety for all purposes. The variable regions of each light / heavy chain pair form the antibody binding site.

  Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

  All chains exhibit the same overall structure of relatively conserved framework regions (FR) joined by three hypervariable regions (also called CDRs). Each pair of double stranded CDRs aligns beside the framework region and allows binding to a specific epitope. Both the N-terminal to C-terminal ends of the light and heavy chains contain the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The arrangement of amino acids for each region was determined by KabatSequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lek. According to the definition of Chothia et al. Nature 342: 878-883 (1989).

  A bispecific antibody or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab 'fragments. See, for example, Songsivillai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol. 148: 1547-1553 (1992). Bispecific antibodies do not exist in the form of fragments having a single binding site (eg, Fab, Fab ', and Fv).

  While the VH or VL region can be used alone to bind to the antigen, generally the VH region is paired with the VL region to provide an antigen binding site for the antibody. The VH region (see Table 2) can be paired with the VL region (see Table 2) such that the antigen binding site of the antibody is formed to include both the VH and VL regions.

Human antibodies and humanized human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and / or constant regions. The presence of such mouse or rat derived proteins can lead to rapid removal of the antibody or can lead to the generation of an immune response to the antibody by the patient. In order to avoid the use of antibodies derived from mice or rats, the functional human antibody locus should be placed in rodents, other mammals or so that rodents, other mammals or animals can produce fully human antibodies. Through introduction into animals, fully human antibodies can be produced.

  One method for the production of fully human antibodies is the XenoMouse® of mice engineered to contain germline-sized fragments of the human heavy chain locus and the kappa light chain locus of size less than 1000 kb. ) Using the system. See Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobobits J. Exp. Med. 188: 483-495 (1998). The XenoMouse® line is available from Amgen (Fremont, California, USA).

  Such mice can produce human immunoglobulin molecules and antibodies, and cannot produce mouse immunoglobulin molecules and antibodies. The technology utilized to accomplish this preparation is described in US patent application Ser. No. 08 / 759,620 filed Dec. 3, 1996 and WO 98/24893 and 2000 published Jun. 11, 1998. Which are disclosed in WO 00/76310, published December 21, 2003, the disclosures of which are incorporated herein by reference. See also Mendez et al Nature Genetics 15: 146-156 (1997), the disclosure of which is hereby incorporated by reference.

  The generation of the XenoMouse® strain of mice is described in US patent application Ser. No. 07 / 466,008 filed on Jan. 12, 1990, 07 / 610,515 filed Nov. 8, 1990. No. 07 / 919,297 filed July 24, 1992, 07 / 922,649 filed July 30, 1992, filed March 15, 1993 08 / 031,801, 08 / 112,848 filed on August 27, 1993, 08 / 234,145 filed April 28, 1994, 1995 1 No. 08 / 376,279, filed on May 20, 08 / 430,938, filed April 27, 1995, 08/464, filed Jun. 5, 1995 , 584, 1995 No. 08 / 464,582, filed Jun. 5, 1995, No. 08 / 463,191, filed Jun. 5, 1995, No. 08, filed Jun. 5, 1995 No. 462,837, 08 / 086,853 filed on June 5, 1995, 08 / 486,857 filed Jun. 5, 1995, June 5, 1995 No. 08 / 486,859, filed Jun. 5, 1995, No. 08 / 462,513, filed Oct. 2, 1996, No. 08 / 724,752. No. 08 / 759,620 filed on Dec. 3, 1996, U.S. Patent Publication No. 2003/0093820, filed Nov. 30, 2001, U.S. Pat. No. 6,162,963, ibid. No. 6,150,584, No. 6,114 598, 6,075,181 and 5,939,598 as well as Japanese Patent Nos. 3,068,180, 3,068,506 and 3,068,507, and are further described in detail. ing. European Patent No. 0 463 151, published on June 12, 1996, WO 94/02602 published on February 3, 1994, WO 96/34096 published on October 31, 1996, 1998 See also WO 98/24893 published on June 11, 2000 and WO 00/76310 published on December 21, 2000. The disclosures of each of the patents, patent applications, and references cited above are hereby incorporated by reference in their entirety.

In one alternative approach, others including GenPharm International have utilized a “minilocus” approach. The minilocus approach mimics an exogenous Ig locus by including multiple pieces (individual genes) from the Ig locus. Thus, one or more V _H genes, one or more _DH genes, one or more J _H genes, a μ constant region, and usually a second constant region (preferably a γ constant region) are animals. Formed in a construct for insertion into This approach is described in U.S. Pat. No. 5,545,807 by Suranietal, and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126 by Lonberg and Kay, respectively. No. 5,633,425, No. 5,661,016, No. 5,770,429, No. 5,789,650, No. 5,814,318, No. 5 , 877,397, 5,874,299, and 6,255,458, US Pat. Nos. 5,591,669 and 6,023.010 by Krimpenfort and Berns, Bernset al. U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215, and Choi and D U.S. Patent No. 5,643,763 to nn, and GenPharm International, U.S. Patent Application No. 07 / 574,748, filed Aug. 29, 1990, No. 07, filed Aug. 31, 1990. No. 575,962, No. 07 / 810,279 filed Dec. 17, 1991, No. 07 / 853,408 filed Mar. 18, 1992, Jun. 23, 1992. No. 07 / 904,068, filed on Dec. 16, 1992, No. 07 / 990,860 filed on Dec. 16, 1992, and No. 08 / 053,131 filed on Apr. 26, 1993. No. 08 / 096,762 filed on Jul. 22, 1993, No. 08 / 155,301 filed Nov. 18, 1993, No. 12/1993. No. 08 / 161,739 filed on the 3rd, 08 / 165,699 filed on the 10th December 1993, 08/209, filed on the 9th March 1994, 741, the disclosures of which are incorporated herein by reference. European Patent No. 0 546 073, WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97 / See also 13852, and WO 98/24884 and US Pat. No. 5,981,175, which are hereby incorporated by reference in their entirety. Furthermore, Taylor et al, 1992, Chen et al, 1993, Tuaillon et al, 1993, Choi et al, 1993, Lonberg et al, (1994), Taylor et al, (1994), and Tuaillon et al, (1995). , Fishwild et al, (1996), which are incorporated herein by reference in their entirety.

  Kirin also showed the production of human antibodies from mice into which a large part of the chromosome or the entire chromosome has been introduced by microcell fusion. See European Patent Applications Nos. 773 288 and 843 961, the disclosures of which are hereby incorporated by reference. In addition, KM ™ mice were generated as a result of cross-breeding Kirin Tc mice with Medarex minilocus (Humab) mice. These mice carry the Kirin mouse human IgH transchromosome and the Genpharm mouse kappa chain transgene (Ishida et al, Cloning Stem Cells, (2002) 4: 91-102).

  Human antibodies can also be generated by in vitro methods. Suitable examples include, but are not limited to, phage display (MedImmune, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed), ribosome display (MedImmune), yeast display, and the like.

Antibody Preparation The antibodies described herein were prepared using XenoMouse® technology as follows. Such mice can produce human immunoglobulin molecules and antibodies, and cannot produce mouse immunoglobulin molecules and antibodies. The techniques utilized to accomplish this preparation are disclosed in the patents, patent applications, and references disclosed in the background section of this specification. However, in particular, a preferred embodiment of transgenic production of mice and antibodies by that technique is disclosed in US patent application Ser. No. 08 / 759,620 filed Dec. 3, 1996 and published Jun. 11, 1998. International Publication No. WO 98/24893 and WO 00/76310 published Dec. 21, 2000, the disclosures of which are incorporated herein by reference. Mendez et al. See also Nature Genetics 15: 146-156 (1997), the disclosure of which is incorporated herein by reference.

  Using this technique, fully human monoclonal antibodies against various antigens have been produced. Basically, a mouse XenoMouse (registered trademark) strain is immunized with a target antigen (eg, DLL4), lymphocytes (B cells, etc.) are recovered from a hyperimmune mouse, and the recovered lymphocytes are used as a bone marrow type cell line. To prepare an immortal hybridoma cell line. These hybridoma cell lines are screened to select and identify hybridoma cell lines that produce antibodies specific for the antigen of interest. The present specification provides methods for producing a plurality of hybridoma cell lines that produce antibodies specific for DLL4. In addition, the present specification provides characterization of antibodies produced by such cell lines, including analysis of the nucleotide and amino acid sequences of the heavy and light chains of such antibodies.

  Alternatively, instead of fusing with myeloma cells to produce hybridomas, B cells can be assayed directly. For example, CD19 + B cells can be isolated from hyperimmune XenoMouse® mice to proliferate and differentiate antibody secreting plasma cells. The reactivity of the antibody from the cell supernatant to the DLL4 immunogen is then screened by, for example, ELISA, FACS, or FMAT. This supernatant may also be screened for immunoreactivity to the DLL4 fragment and further mapped with different antibodies that bind to a functional region of interest on DLL4. Antibodies may be screened for other orthologs of DLL4 against other related human DLL4 and rats, mice, and non-human primates (such as cynomolgus monkeys) and finally species cross-reactivity determined. B cells from wells containing the antibody of interest produce hybridomas by various methods (either fused and individual wells or pooled wells, or by EBV infection or by transfection with a known immortalizing gene. May be immortalized and then placed in a suitable medium. Alternatively, single antibody-secreting plasma cells with the desired specificity are then isolated using a DLL4-specific hemolytic plaque assay (eg, Babcook et al, Proc. Natl. Acad. Sci. USA 93: 7843-48 ( 1996)). Cells targeted for lysis are preferably sheep red blood cells (SRBC) coated with DLL4 antigen.

  In the presence of a B cell culture containing plasma cells and complement that secrete the immunoglobulin of interest, plaque formation indicates specific DLL4 mediated lysis of sheep red blood cells surrounding the subject plasma cells. A single antigen-specific plasma cell at the plaque center can be isolated, and the genetic information encoding the specificity of the antibody is isolated from a single plasma cell. After using reverse transcription, PCR can be performed (RT-PCR) to clone DNA encoding the heavy and light chain variable regions of the antibody. Such cloned DNA can then be further inserted into a suitable expression vector, preferably a vector cassette (such as pcDNA), more preferably a pcDNA vector comprising immunoglobulin heavy and light chain constant regions. This produced vector is then transfected into host cells (eg, HEK293 cells, CHO cells) and modified to be suitable for induction of transcription, selection of transformants, or amplification of the gene encoding the desired sequence. Can be cultured in a nutrient medium.

  As will be appreciated, antibodies that specifically bind to DLL4 can be expressed in cell lines other than hybridoma cell lines. In order to transform suitable mammalian host cells, sequences encoding particular antibodies can be used. Transformation is by any known method for introducing a polynucleotide into a host cell, including encapsulation of the polynucleotide in a virus (or in a viral vector) and transduction of the host cell by the virus (or vector), Or US Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (these patents are incorporated herein by reference). This is possible by transfection procedures known in the art as exemplified in). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotides into liposomes. (S) encapsulation and direct microinjection of DNA into the nucleus.

  Mammalian cell lines that can be used as expression hosts are well known in the art, and many immortal cell lines (Chinese hamster ovary (CHO) cells, available from the American Type Culture Collection (ATCC), Including, but not limited to, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), human epithelial kidney 293 cells, and several other cell lines Not). Through the determination of which cell lines have high expression values and produce antibodies with constitutive DLL4 binding properties, particularly preferred cell lines are selected.

  Cell fusion technology prepares a myeloma, CHO cell or other cell line with a heavy chain with any desired isotype, and another myeloma, CHO cell or other cell line with a light chain. . Such cells can then be fused to isolate cell lines expressing intact antibodies.

  Thus, antibody candidates are usually produced to meet the desired “structural” properties as discussed above, and thus typically provide antibody candidates with at least some desired “functional” properties via isotype switches. can do.

Therapeutic Administration and Formulations Embodiments of the invention include sterile pharmaceutical formulations of anti-DLL4 antibodies that are useful in the treatment of disease. Such formulations inhibit the binding of native DLL4 to Notch1 or Notch4 receptors, thereby effectively treating, for example, symptoms of abnormally elevated serum or tissue DLL4 expression. The anti-DLL4 antibody preferably has a suitable affinity that strongly inhibits the binding of native DLL4 to Notch1 or Notch4 receptor, preferably with a suitable duration of action, and is administered with low frequency administration to humans. Make it possible. Prolonged duration of action allows less frequent and more convenient dosing regimens by alternative parenteral routes (such as subcutaneous or intramuscular injection).

  Sterile formulations can be made before or after antibody lyophilization and reconstitution, for example, by filtration through sterile filtration membranes. This antibody is usually stored in lyophilized form or in solution. The therapeutic antibody composition is usually placed in a vial with a sterile access port (eg, an intravenous solution bag) or an adapter (such as a stopper that can be pierced by a hypodermic needle) that allows for the recovery of the formulation. It is done.

  The route of antibody administration is known in the art (eg, intravenous or intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intrathecal injection or infusion, inhalation or intralesional route, direct injection at the tumor site. Or the following sustained release system). The antibody is preferably administered continuously by infusion or bolus injection.

  The effective amount of antibody used therapeutically will vary depending on, for example, the therapeutic purpose, route of administration, and patient condition. Therefore, it is preferable that the therapist titrate the dose and change the administration route as required to obtain the optimal therapeutic effect. Typically, the clinician will administer antibody until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays or by the assays described herein.

  The antibodies described herein can be prepared in a mixture with a pharmaceutically acceptable carrier. The therapeutic composition can be administered intravenously or via the nose or lung, preferably as a liquid or powder aerosol (lyophilized). This composition may be administered parenterally or subcutaneously as desired. When administered systemically, the therapeutic composition should be sterile, pyrogen-free, and in a parenterally acceptable solution with respect to pH, isotonicity, and stability. These conditions are known to those skilled in the art. Briefly, dosage formulations of the compounds described herein are prepared for storage or administration by mixing a pharmaceutically acceptable carrier, excipient, or stabilizer with a compound having the desired purity. Is done. Such materials are non-toxic to recipients at the dosages and concentrations used, buffers (such as Tris-HCl, phosphate, citrate, acetate and other organic acid salts); antioxidants (such as ascorbic acid) ); Low molecular weight (less than about 10 residues) peptides (such as polyarginine), proteins (such as serum albumin, gelatin, or immunoglobulin); hydrophilic polymers (such as polyvinylpyrrolidinone); amino acids (glycine, glutamic acid, aspartic acid, or Monosaccharides, disaccharides, and other carbohydrates (including cellulose or its derivatives, glucose, mannose, or dextrin); chelating agents (such as EDTA); sugar alcohols (such as mannitol or sorbitol); counterions (sodium) Etc.) and / or non-io Sex surfactant containing (TWEEN, PLURONICS or polyethylene glycol, etc.).

Sterile compositions for ^{injection, Remington: The Science and Practice of} Pharmacy can be formulated according to conventional pharmaceutical practice as described in (. 20 th ed, Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a pharmaceutically acceptable carrier (such as water or a natural vegetable oil such as sesame oil, peanut oil, or cottonseed oil, or a synthetic fat vehicle such as ethyl oleate) may be desired. . Buffers, preservatives, antioxidants, etc. can be incorporated according to accepted pharmaceutical practice.

  Suitable examples of sustained release preparations include solid hydrophobic polymer semipermeable substrates containing polypeptides, which are in the form of shaped articles, films or microcapsules. Examples of sustained release substrates include those described by Langer et al. , J .; Biomed Mater. Res. (1981) 15: 167-277 and Langer, Chem. Tech. (1982) 12: 98-105, polyesters, hydrogels (eg, poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919). , European Patent No. 58,481), copolymers of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22: 547-556), non-degradable ethylene-vinyl acetate ( Langer et al.), Degradable lactic acid-glycolic acid copolymers (such as LUPRON Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D-(−) -3-hydroxybutyric acid European Patent No. 133,988) can be mentioned.

  While polymers (such as ethylene-vinyl acetate and lactic acid-glycolic acid) can release molecules over 100 days, certain hydrogels release proteins in a shorter period of time. If encapsulated proteins remain in the body for an extended period of time, they may denature or aggregate as a result of exposure to moisture at 37 ° C., losing biological activity and altering immunogenicity. Rational strategies for protein stabilization can be devised depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via disulfide interconversion, modification of sulfhydryl residues, lyophilization from acidic solution, control of moisture content, appropriate Stabilization can be achieved through the use of additives and the generation of specific polymer matrix compositions.

  Sustained release compositions also include preparations of antibody crystals suspended in a suitable formulation that can maintain the crystals in suspension. These preparations can provide a sustained release effect upon subcutaneous or intraperitoneal injection. Other compositions also include antibodies encapsulated in liposomes. Liposomes containing such antibodies are basically known in the manner described in German Patent 3,218,121; Epstein et al. , Proc. Natl. Acad. Sci. USA, (1985) 82: 3688-3692; Hwang et al. , Proc. Natl. Acad. Sci. USA, (1980) 77: 4030-4034; European Patent 52,322; European Patent 36,676; European Patent 88,046; European Patent 143,949; 142,641; Prepared according to Japanese Patent Application No. 83-118008; US Pat. Nos. 4,485,045 and 4,544,545; and European Patent No. 102,324.

  The dosage of the antibody formulation administered to the patient depends on various factors known to alter drug action (severity and type of disease, body weight, gender, diet, time and route of administration, other agents and other Determined by the attending physician, taking into account relevant clinical factors). A therapeutically effective amount may be determined by either in vitro or in vivo methods.

  The effective amount of a therapeutically used antibody described herein will vary depending, for example, upon the therapeutic purpose, route of administration, and patient condition. Therefore, it is preferable that the therapist titrate the dose and change the administration route as required to obtain the optimal therapeutic effect. Typical daily dosages are about 0.0001 mg / kg, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 10 mg / kg up to 100 mg / kg per patient body weight, The range may be up to 1000 mg / kg, 10,000 mg / kg or more, and varies depending on the above-described factors. Dose is 0.0001 mg / kg to 20 mg / kg, 0.0001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 5 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg per patient body weight / Kg, 0.0001 mg / kg to 0.75 mg / kg, 0.0001 mg / kg to 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0 It may be 0.0001-0.10 mg / kg, 0.001-0.5 mg / kg, 0.01-0.25 mg / kg or 0.01-0.10 mg / kg, depending on the above factors. Typically, the clinician will administer the therapeutic antibody until the dosage reaches the desired effect. The progress of this therapy is easily monitored by conventional assays or as described herein.

  The administration of the antibody of the present invention may be repeated, and this administration is at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months Or an interval of at least 6 months.

  The therapeutic entities according to the compositions and methods herein are administered in combination with suitable carriers, excipients, and other agents incorporated into the formulation to obtain improved transfer, delivery, tolerability, etc. It will be understood. These formulations include, for example, powders, plasters, ointments, jellies, waxes, oils, lipids, lipids including vesicles (such as Lipofectin ™) (cationic or anionic), DNA conjugates, Anhydrous absorbent muds, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing carbowax. Any of the above mixtures may be suitable for treatment and therapy according to the present invention if the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and well tolerated by the route of administration. Baldrick P.M. “Pharmaceutical exclusive development: the need for preclinical guidance.” Regul. Toxicol. Pharmacol. 32 (2): 210-8 (2000), Wang W., et al. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. et al. Pharm. 203 (1-2): 1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some developing concepts.” J Pharm Sci. 89 (8): 967-78 (2000), Powell et al. “Compendium of exclusives for parental formulations” PDA J Pharm Sci Technol. 52: 238-311 (1998) and additional information related to formulations, excipients and carriers well known to the pharmaceutical chemist cited therein.

Other Therapeutic Designs and Productions Based on the present invention and the activity of the antibodies generated and characterized herein for DLL4, the design of other therapeutic modalities other than the antibody portion is facilitated. Such modalities include advanced antibody therapies (such as bispecific antibodies, immunotoxins, and radiolabeled therapies), single domain antibodies, antibody fragments (such as Fab, Fab ′, F (ab ′) ₂ , Fv or dAb), Production of peptide therapies, DLL4 binding regions in new scaffolds, gene therapy, including but not limited to intracellular antibodies, antisense therapies, and small molecules.

  Antigen binding sites can be determined using CDR sequences on non-antibody scaffold proteins (such as fibronectin or cytochrome B) (Haan & Maggos (2004) BioCentury, 12 (5): A1-A6; Koide et al. (1998) Journal of Molecular Biology, 284: 1141-1151; Nygren et al. (1997) Current Opinion in Structural Biology, 7: 463-469), or randomizing or changing the amino acid residues of the loops within the scaffold protein to the desired target It can be provided by conferring binding specificity. Scaffolds for genetic engineering of new binding sites of proteins are described in Nygren et al. (Nygren et al. (1997) Current Opinion in Structural Biology, 7: 463-469). Antibody mimetic scaffold proteins are disclosed in WO 0034784, which is hereby incorporated by reference in its entirety, and we have a protein comprising a fibronectin type III region with at least one randomized loop (Antibody mimetics) are described. Any region member of the immunoglobulin gene superfamily may provide a suitable scaffold for transplanting one or more CDRs (eg, a group of HCDRs). This scaffold may be a human or non-human protein. The advantages of non-antibody scaffold proteins may provide antigen binding sites in the molecular scaffold that are smaller and / or easier to manufacture than at least some antibody molecules. Small size binding members confer useful physiological properties, such as the ability to enter cells, penetrate deeply into tissues, reach targets in other structures, or bind within the protein cavity of target antigens obtain. The use of antigen binding sites in non-antibody scaffold proteins is reviewed in Wess, 2004 (Wess, L. In: BioCentury, The Bernstein Report on BioBusiness, 12 (42), A1-A7, 2004). Typical are proteins having a stable backbone and one or more variable loops in which the amino acid sequence of the loop (s) is specifically or randomly mutated to target antigen Forms an antigen binding site that binds to Such proteins include protein A from S. aureus, transferrin, albumin, tetranectin, fibronectin (eg, 10th fibronectin type III region), lipocalin and γ-crystallin and other Affilin ™ scaffolds (Scil Proteins). An IgG binding region can be mentioned. Examples of other approaches include synthetic “microbodies” based on cyclotide-small proteins with intramolecular disulfide bonds, microproteins (Versabodies ™, Amunix) and ankyrin repeat proteins (DARPins, Molecular Partners).

  In addition to antibody sequences and / or antigen binding sites, the targeted binding agents according to the present invention may contain other amino acids, such as peptide or polypeptide forms, folded regions, etc., or separate molecules in addition to antigen binding capacity. Functional properties of The targeted binding agents of the invention may have a detectable label or may be conjugated to a toxin or target moiety or enzyme (eg, via a peptidyl bond or linker). For example, the targeted binding agent can include a catalytic site (eg, within the enzyme region) as well as an antigen binding site (antigen binding site that binds to the antigen), whereby the catalytic site can target the antigen. This catalytic site may inhibit the biological function of the antigen, for example by cleavage.

  In connection with the production of advanced antibody therapeutics, complement binding is a desirable property, eg avoiding complement dependence for cell death through the use of bispecific antibodies, immunotoxins, or radiolabels It may be possible to do.

  For example, (i) one specific for DLL4 and the other specific for a second molecule bound to each other, (ii) specific for DLL4 Produce a bispecific antibody comprising a single antibody having a single chain and a second chain that is specific for a second molecule, or (iii) a single chain antibody having specificity for DLL4 and other molecules it can. Such bispecific antibodies are described, for example, in connection with (i) and (ii) (see, eg, Fanger et al. Immuno Methods 4: 72-81 (1994) and the above Wright and Harris), and (Iii) (see, eg, Traunecker et al. Int. J. Cancer (Suppl.) 7: 51-52 (1992)). In each instance, the second specificity is CD16 or CD64 (see, eg, Deo et al. Immunol. Today 18: 127 (1997)) or CD89 (eg, Valerius et al. Blood 90: 4485-4492 ( (1997))) can be generated into heavy chain activated receptors including, but not limited to.

  An antibody can also be modified to act as an immunotoxin using techniques well known in the art. See, for example, Vitetta Immunol Today 14: 252 (1993). See also US Pat. No. 5,194,594. In connection with the preparation of radiolabeled antibodies, such modified antibodies can also be readily prepared using techniques well known in the art. For example, Junghans et al. See in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds, Lippincott Raven (1996)). U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE35,500), 5,648,471 See also, and 5,697,902. Each immunotoxin or radiolabeled molecule may kill cells that express the desired multilinked enzyme subunit oligomerization region.

  When an antibody is conjugated to an agent (eg, a radioisotope, pharmaceutical composition, or toxin), the agent is an anti-mitotic agent, alkylating agent, antimetabolite, anti-angiogenic agent, apoptotic agent, alkaloid agent, It is intended to possess pharmaceutical properties selected from the group of COX-2 agents, and antibiotics and combinations thereof. Drugs are nitrogen mustard, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antimetabolites, antibiotics, enzymes , Epipodophyllotoxin, platinum coordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenal cortex inhibitors, antagonists, endostatin, taxol, camptothecin, oxaliplatin, doxorubicin and analogs thereof, and combinations thereof You can choose from the group of

  Examples of toxins further include gelonin, Pseudomonas aeruginosa exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNaseI, staphylococcal enterotoxin-A, pokeweed antiviral Examples include proteins, gelonin, Pseudomonas aeruginosa endotoxin, enediyne family members of the molecule (such as calicheamicin and esperamycin), and derivatives, combinations and modifications thereof. Chemical toxins include duocarmycin (see, eg, US Pat. Nos. 5,703,080 and 4,923,990), methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, It can also be selected from the group consisting of mitomycin C, cis-platinum, etoposide, bleomycin and 5-fluorouracil. Examples of chemotherapeutic agents include adriamycin, doxorubicin, 5-fluorouracil, cytosine arabinoside (Ara-C), cyclophosphamide, thiotepa, taxotere (docetaxel), busulfan, cytoxin, taxol, methotrexate, cisplatin (Cisplatin), melphalan, vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamicin No. 4,675,187), melphalan, and other related nitrogen mustards. . Suitable toxins and chemotherapeutic agents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995) and Goodman And Gilman's The Pharmaceuticals Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985). Other suitable toxins and / or chemotherapeutic agents are known to those skilled in the art.

  Examples of radioisotopes include γ-emitters, positron emitters and X-ray emitters that can be used for localization and / or therapy, and β- and α-emitters that can be used for therapy. . Radioisotopes previously described as useful for diagnosis, prognosis and staging are also useful for therapy.

  Non-limiting examples of anti-cancer or anti-leukemic agents include anthracyclines such as doxorubicin (adriamycin), daunorubicin (daunomycin), idarubicin, detorubicin, carminomycin, epirubicin, esorubicin, and morpholinos and substituted derivatives, combinations thereof And modifications. Representative pharmaceuticals include cisplatin, taxol, calicheamicin, vincristine, cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin, idarubicin, fludarabine, chlorambucil, interferon alpha, hydroxyurea, temozolomide, thalidomide, and Bleomycin, and derivatives, combinations and modifications thereof are mentioned. Preferably, the anti-cancer or anti-leukemia agent is doxorubicin, morpholino doxorubicin, or morpholino daunorubicin.

  The antibody of the present invention also includes an antibody whose half-life (eg, serum half-life) in a mammal (preferably human) is longer than that of an unmodified antibody. The antibody has a half-life of more than about 15 days, more than about 20 days, more than about 25 days, more than about 30 days, more than about 35 days, more than about 40 days, more than about 45 days, more than about 2 months, more than about 3 months , Greater than about 4 months, or greater than about 5 months. Prolonged half-life of the antibody or fragment thereof of the present invention in a mammal (preferably human) leads to a higher serum titer of said antibody or antibody fragment in the mammal, whereby administration of said antibody or antibody fragment Reduce the frequency and / or decrease the concentration of the antibody or antibody fragment administered. Antibodies or fragments thereof having a prolonged in vivo half-life can be produced by techniques known to those skilled in the art. For example, an antibody or fragment thereof having a prolonged in vivo half-life is produced by modifying (eg, substituting, deleting, or adding) amino acid residues that have been identified as involved in the interaction between the Fc region and the FcRn receptor. (See, eg, International Publication Nos. WO 97/34631 and WO 02/060919, which are incorporated herein by reference in their entirety). An antibody or fragment thereof having a prolonged in vivo half-life can be produced by binding to the antibody or antibody fragment polymer molecule (such as high molecular weight polyethylene glycol (PEG)). PEG with or without a multifunctional linker via either a site-specific conjugation of PEG to the N or C terminus of the antibody or antibody fragment or an epsilon-amino group present on a lysine residue, Can bind to antibody fragments. Linear or branched polymer derivatization with minimal loss of biological activity is used. This degree of conjugation is closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from antibody-PEG conjugates by, for example, size exclusion or ion exchange chromatography.

  As will be appreciated by those skilled in the art, in the above embodiments, affinity values are important, but other factors may be equally or more important depending on the particular function of the antibody. For example, in immunotoxins (antibody-related toxins), the binding action of the antibody to the target may be useful; however, in some embodiments, the desired end result is internalization of the toxin into the cell. Thus, antibodies with high percent internalization may be desired in these situations. Thus, in one embodiment, antibodies with high internalization efficiency are contemplated. High efficiency of internalization can be measured as a percentage of internal antibody and can range from low to 100%. For example, in various embodiments, 0.1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-99, and 99-100% can be highly efficient. As will be appreciated by those skilled in the art, the desired efficiency may vary in different embodiments, for example, related agents, the amount of antibody that can be administered to the site, side effects of antibody-drug conjugates, the type of problem to be treated (eg, , Cancer type) and severity.

  In other embodiments, the antibodies disclosed herein provide a detection assay kit for DLL4 expression in mammalian tissue or cells to screen for diseases or disorders associated with altered DLL4 expression. The kit includes an antibody that binds to DLL4 and, if present, means for indicating an antibody antigen response.

Combinations Targeted binding agents or antibodies as defined herein may be applied as monotherapy or may involve conventional surgery or radiation therapy or chemotherapy in addition to the compounds of the invention. Such chemotherapy may include one or more of the following types of anti-tumor agents:
(I) alkylating agents (eg cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolomide and nitrosourea) used in medical oncology; For example gemcitabine and folic acid antagonists (such as fluoropyrimidines such as 5-fluorouracil and tegaflu), raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumor antibiotics (eg, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, Anthracyclines such as idarubicin, mitomycin-C, dactinomycin and mitramycin); anti-mitotic agents (eg vincristine, Vinca alkaloids such as blastine, vindesine and vinorelbine and taxoids such as taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (eg epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan and camptothecin) Other anti-proliferative / anti-neoplastic agents and combinations thereof;
(Ii) antiestrogens (eg tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxifene), antiandrogens (eg bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (eg goserelin) , Leuprorelin and buserelin), progestogens (eg megestrol acetate), aromatase inhibitors (eg as anastrozole, letrozole, borazole and exemestane) and cells such as 5α-reductase inhibitors such as finasteride Growth inhibitor;
(Iii) anti-invasive agents (eg 4- (6-chloro-2,3-methylenedioxyanilino) -7- [2- (4-methylpiperazin-1-yl) ethoxy] -5-tetrahydropyran-4 -Iloxyquinazoline (AZD0530; International Publication No. WO 01/94341) and N- (2-chloro-6-methylphenyl) -2- {6- [4- (2-hydroxyethyl) piperazin-1-yl] -2 C-Src kinase family inhibitors such as methylpyrimidin-4-ylamino} thiazole-5-carboxamide (dasatinib, BMS-354825; J. Med. Chem, 2004, 47, 6658-6661), as well as marimastat Metalloproteinase inhibitors, urokinase plasminogen activator receptor function inhibitors or , Cathepsin inhibitors, serine protease inhibitors, for example matriptase, hepsin, urokinase, heparanase inhibitors);
(Iv) cytotoxic agents (such as fludarabine, 2-chlorodeoxyadenosine, chlorambucil or doxorubicin) and combinations thereof (fludarabine + cyclophosphamide, CVP: cyclophosphamide + vincristin + prednisone, ACVBP: doxorubicin + cyclophosphine) Famide + vindesine + bleomycin + prednisone, CHOP: cyclophosphamide + doxorubicin + vincristine + prednisone, CNOP: cyclophosphamide + mitoxantrone + vincristine + prednisone, m-BACOD: methotrexate + bleomycin + doxorubicin + cyclophosphine Do + vincristine + dexamethasone + leucovorin, MACOP-B: methotrexate + doxorubicin + cyclophosphine + Vincristine + prednisone fixed dose + bleomycin + leucovorin, or ProMACE CytaBOM,: prednisone + doxorubicin + cyclophosphamide + etoposide + cytarabine + bleomycin + vincristine + methotrexate + leucovorin, etc.).

(V) inhibitors of growth factor function, eg, such inhibitors include growth factor antibodies and growth factor receptor antibodies (eg, anti-erbB2 antibody trastuzumab [Herceptin ™], anti-EGFR antibody panitumumab, anti-erbB1 antibody cetuximab [Arbitux (Erbitux), C225] and Stern et al. Critical reviews in oncology / haeology, 2005, Vol. 54, pp11-29), and as such inhibitors. Tyrosine kinase inhibitors such as epidermal growth factor family inhibitors (eg N- (3-chloro-4-fluorophenyl) -7-methoxy-6- (3-morpholinopropoxy) quinazoline-4-amino (Gefitinib, ZD1839), N- (3-ethynylphenyl) -6,7-bis (2-methoxyethoxy) quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamide-N- (3-chloro EGFR family tyrosine kinase inhibitors such as -4-fluorophenyl) -7- (3-morpholinopropoxy) -quinazoline-4-amine (CI1033), erbB2 tyrosine kinase inhibitors (such as lapatinib), hepatocyte growth factor Family inhibitors, platelet derived growth factor family inhibitors (such as imatinib), serine / threonine kinase inhibitors (eg Ras / Raf signaling inhibitors (eg farnesyl transferase inhibitors such as sorafenib (BAY 43-9006)), MEK and / or Or Inhibitors of cell signaling through AKT kinase, hepatocyte growth factor family inhibitors, c-kit inhibitors, abl kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors, aurora kinase inhibitors (eg AZD1152) , PH 733358, VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459), cyclin dependent kinase inhibitors (such as CDK2 and / or CDK4 inhibitors), and survival signaling protein inhibitors (Bcl-2) , Bcl-XL such as ABT-737);
(Vi) anti-angiogenic agents such as those that inhibit the effects of vascular endothelial growth factor [eg anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin ™), angiopoietin-2 inhibitor, and VEGF receptor tyrosine kinase Inhibitors, for example 4- (4-bromo-2-fluoroanilino) -6-methoxy-7- (1-methylpiperidin-4-ylmethoxy) quinazoline (ZD6474; Example 2 in WO 01/32651), 4 -(4-Fluoro-2-methylindol-5-yloxy) -6-methoxy-7- (3-pyrrolidin-1-ylpropoxy) quinazoline (AZD2171; Example 240 in WO 00/47212), batalanib (PTK787) WO 98/35985) and SU11248 (sunitinib; WO 01/608); 4, Sutent (TM), sorafenib (Nexavar (TM)), International Publication WO 97/22596, WO 97/30035, WO 97/32856, WO 98/13354, WO 00/47212 and Compounds disclosed in WO 01/32651 and compounds acting by other mechanisms (eg linomides, integrin αvβ3 function inhibitors and angiostatin)] or colony stimulating factor 1 (CSF1) or CSF1 receptor;
(Vii) Vascular injury such as combretastatin A4 and compounds disclosed in WO99 / 02166, WO00 / 40529, WO00 / 41669, WO01 / 92224, WO02 / 04434 and WO02 / 08213 Sex drugs;
(Viii) antisense therapy (e.g., directed to targets listed above G-3139 (Genasense), anti-bcl2 antisense, etc.);
(Ix) Abnormal gene replacement approaches such as abnormal p53 or abnormal BRCA1 or BRCA2, GDEPT (gene-directed enzyme prodrug therapy) approaches such as those using cytosine deaminase, thymidine kinase or bacterial nitroreductase, and multiple Gene therapy approaches including approaches to increase patient tolerance to chemotherapy or radiation therapy such as drug resistant gene therapy; and (x) treatment with eg alemtuzumab (campath-1H ™), CD52 oriented Such as treatment with monoclonal antibodies or CD22 directed antibodies, transfection with cytokines (such as interleukin 2, interleukin 4 or granulocyte macrophage colony stimulating factor) In vitro and in vivo approaches to increase the immunogenicity of tumor cells in patients, approaches to reduce T cell anergy (such as monoclonal antibody therapy that inhibits CTLA-4 function), transfections such as cytokine transfected dendritic cells Immunotherapy approaches including approaches using immune cells, approaches using cytokine transfected tumor cell lines and approaches using anti-idiotypic antibodies.

  (Xi) Proteolytic inhibitors such as proteasome inhibitors such as velcade (bortezomib).

  (Xii) peptides or proteins (antibodies or lysis) that either sequester receptor ligands, block receptor-bound ligands or decrease receptor signaling (eg, due to increased receptor degradation or decreased expression values) Biotherapeutic therapeutic approaches using extra-receptor domain structures).

  In one embodiment, an anti-tumor treatment as defined herein is a treatment with an anti-proliferative / anti-neoplastic agent and combinations thereof used in medical oncology (alkyl) in addition to the compounds of the invention. Agents (eg cisplatin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, temozolomide and nitrosourea); antimetabolites (eg gemcitabine and folate antagonists (5-fluorouracil and tegafur) Such as fluoropyrimidine, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea; antitumor antibiotics (eg, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin) , Mitomycin-C, anthracyclines such as dactinomycin and mitramycin); anti-mitotic agents (eg vinca alkaloids such as vincristine, vinblastine, vindesine and vinorelbine and taxoids such as taxol and taxotere and polokinase inhibitors) And topoisomerase inhibitors (eg, epipodophyllotoxins such as etoposide and teniposide, amsacrine, topotecan and camptothecin) and the like.

  In one embodiment, an anti-tumor treatment as defined herein may include treatment with gemcitabine in addition to a compound of the invention.

  Such joint treatment can be accomplished by simultaneous, sequential or divided dose methods of the individual components of the treatment. Such combinations of products use the dosage range of the compounds of the invention as described herein above, or a pharmaceutically acceptable salt thereof, as well as other pharmaceutically active agents in the approved dosage range.

  The following examples (including the experiments performed and results achieved) are provided for illustrative purposes only and are not intended to limit the teachings herein.

Immunization and titering
The extracellular region of human DLL4 (amino acids 1-524) transiently expressed in immunogen Chinese hamster ovary (CHO) cells and recombinant human DLL4 were used as antigens for immunization. For generation of CHO transfectants, human full-length DLL4-cDNA (Yoneya et al., 2001, J. Biochem., 129, 27-34) was inserted into pcDNA3.1 vector and CHO cells (American Type). Lipofected in Tissue Collection, catalog # CCL-61). Expression of human DLL4 on the cell surface in an amount suitable for the purpose of immunization was confirmed by fluorescence activated cell sorting (FACS) analysis. The extracellular region of human DLL4 was subcloned from full-length DLL4 using the forward 5′-AAGCTGGCTAGCGGCAATGGCCGCACGCGCCCGGGAG primer and the reverse 5′-CAGCCTCGAGCGGCCCCCCAGGGGGAAGCTGGGCGGCAAGC primer. The PCR product was purified and ligated into an Invitrogen pSecTag expression vector. This clone was then transfected into 293T cells using 293fectin transfection reagent. After 7 days, the cell supernatant containing the target protein was collected and passed through a pre-equilibrated HisTrap column (GE Healthcare, catalog # 17-5247) overnight. The column is washed with a binding buffer containing 20 mM sodium phosphate, 500 mM sodium chloride and 5 mM imidazole, pH 7.4, and the his tag protein is buffered with 20 mM sodium phosphate, 500 mM sodium chloride and 500 mM imidazole, pH 7.4. Elute with liquid. The protein sample is dialyzed against binding buffer for 1 hour at 4 ° C. and further dialyzed against PBS, pH 7.4 for 2 hours, followed by filter sterilization, SDS-PAGE quantification and purity evaluation, followed by Gelcode (Pierce, catalog # 24950).

Immunization Using either the extracellular region of DLL4 as described in Example 1 or CHO cells overexpressing recombinant human DLL4, XenoMouse® (XenoMouse strains: XMG2 (IgG2κ / λ) and XMG4 (IgG4κ / λ) Amgen, Inc. Vancouver, British Columbia, Canada) Monoclonal antibodies against DLL4 were made by immunizing mice sequentially. In all injections, XenoMouse animals were immunized by intraperitoneal and ridge routes using conventional methods. Adjuvants include Titermax Gold ™ (Sigma, catalog # T2684), aluminum phosphate gel adjuvant, HCL biosector (catalog # 1452-250) and ImmunoEasy mouse adjuvant (qCpG, Qiagen catalog # 303105). For soluble immunogens, a first injection of 10 μg DLL4 extracellular region was performed with Titermax Gold ™ (day 0), along with 5 μg DLL4 extracellular region, aluminum phosphate gel adjuvant and qCpG or aluminum phosphate Alternative boosts were performed using either gel adjuvant and Titremax Gold ™. For cell-based immunogens, the first injection was performed with aluminum phosphate gel adjuvant and 2E6 cells. During the next boost, 1E6 cells were administered using the same adjuvant. For both immunization campaigns, boosts were given on days 2, 6, 10, 16, 23, 30, 37, 44, 50, 64, 71, 75, 89, 104 and 108.

For binding to human and mouse DLL4 expressed in 293T cells using the FLUOROMETRIC microvolume assay technology (FMAT) cell detector (Applied Biosystems) for recovery by titer . The antibody titer against human DLL4 was examined. This analysis indicated that there are mice with titers that appear to be specific for DLL4. Thus, as described in Example 2 below, at the end of the immunization program, 17 mice were selected for recovery and lymphocytes were isolated from the spleen and lymph nodes of the immunized mice.

Lymphocyte collection, B cell isolation, hybridoma fusion and generation Immunized mice were sacrificed by cervical dislocation and draining lymph nodes were collected and stored from each cohort. Lymphoid cells were dissociated by grinding in DMEM to release the cells from the tissue and the cells were suspended in DMEM. B cells were enriched by positive selection using CD19 labeled Dynabeads. Fusion was performed by mixing the wash-concentrated B cells and non-secretory myeloma P3X63Ag8.653 cells (catalog # CRL1580) purchased from ATCC in a 1: 1 ratio (Kearney et al., J. Biol. Immunol. 123, 1979, 1548-1550). The cell mixture was gently pelleted by centrifugation at 800 × g. After complete removal of the supernatant, the cells were treated with 2-4 ml of pronase solution (CalBiochem, catalog # 53702; 0.5 mg / ml in PBS) for only 2 minutes. Thereafter, 3-5 ml of FBS was added to stop the enzyme activity, and an electron cell fusion solution ECFS (0.3 M sucrose, Sigma, catalog # S7903, 0.1 mM magnesium acetate, Sigma, catalog was added to this suspension. The total volume was adjusted to 40 ml using # M2545, 0.1 mM calcium acetate, Sigma, catalog # C4705). After centrifugation, the supernatant was removed and the cells were resuspended with 40 ml ECFS. This washing step was repeated and the cells were resuspended again with ECFS to a concentration of 2E6 cells / ml. Electron-cell fusion was performed using a fusion generator, Model ECM2001, Genetronic, San Diego, California. The fusion chamber size used was 2.0 ml, using the following equipment settings: alignment conditions: voltage: 50 V, time: 50 seconds; membrane breakage: voltage: 3000 V, time: 30 μsec; residence time after fusion: 3 seconds did. After ECF, the cell suspension is carefully removed from the fusion chamber under sterile conditions and the same amount of hybridoma medium (2 mM L-glutamine (Sigma, catalog # G2150), 10 U / ml penicillin / 0.1 mg / ml streptomycin (Sigma, Catalog # P7539) DMEM (JRH Biosciences) supplemented with 1 vial / L OPI (oxaloacetate, pyruvate, bovine insulin; Sigma catalog # O5003) and 10 U / ml recombinant human IL-6 (Boehringer Mannheim, catalog # 1131567) ), 15% FBS (Hyclone)). The cells were incubated at 37 ° C. for 15-30 minutes and then centrifuged at 400 × g for 5 minutes. The cells are gently resuspended in a small amount of hybridoma selection medium (hybridoma medium supplemented with 0.5 × HA (Sigma, Catalog # A9666)) and 5E6 B cells are finally placed in a 96-well plate at 200 μl per well. The amount was further adjusted appropriately with the hybridoma selection medium. The cells were gently mixed and pipetted into 96-well plates to grow. All supernatants were collected from cells potentially producing anti-DLL4 antibodies and then subjected to screening assays as exemplified below.

Binding to cells and binding to human and cynomolgus DLL4 and human JAGGED1 Supernatants collected from harvested cells are examined and 293T cells transiently overexpressing either full-length human or cynomolgus DLL4 or human Jagged1. The binding ability of the secretory antibody was evaluated. A mock transfected 293T cell line was used as a negative control. Cells diluted in PBS containing 2% FBS were seeded in 384 well plates (Corning Costar, catalog # 3712) at a density of 3000 expressing cells and 15000 mock-transfected cells per well. Immediately after plating, 15 or 20 μl / well hybridoma supernatant and 10 μl / well secondary antibody (goat anti-human IgG Fc Cy5, final concentration 750 ng / ml) are added and the plates are incubated at room temperature for 3 hours. Fluorescence was read on an FMAT 8200 instrument (Applied Biosystems). The binding of human Notch1 / Fc chimera (R & D systems, catalog # 3647-TK) diluted 1: 2 from 2.86 μg / ml was used as a positive control for DLL4, and human Notch3 / Fc chimera diluted from 10 μg / ml was used. Used as a positive control for binding to Jagged1. The results of 12 hybridoma supernatants showing the binding of the hybridoma supernatant to human / cynomolgus monkey DLL4 and human Jagged are shown in Table 3.

Inhibition of Notch1-DLL4 receptor-ligand binding Ability to inhibit human Notch1 / Fc binding to human DLL4 transiently overexpressed in 293T cells to determine the relative titer of antibodies, including supernatant Evaluated. Transfected and non-transfected 239T cells were reconstituted with PBS containing 2% FCS, and 5000 transfected cells and 17500 untransfected cells were 384 well tissue culture plates (Corning Costar, catalog #). 3712) wells were seeded in a volume of 20 μl. Subsequently, 20 μl of hybridoma supernatant was added and the plate was incubated for 1 hour at 4 ° C., at which time 20 μl of Alexa-647 labeled human Notch1 / Fc was added at a final concentration of 6.7 ng / ml. After further incubation at 4 ° C. for 3 hours, the amount of Notch1 / Fc binding was measured by reading the fluorescence of each well using an FMAT 8200 instrument (Applied Biosystems). The results for 12 hybridoma supernatants are shown in Table 4. Results as minimal inhibition in the assay as measured by the effect of non-DLL4 binding supernatant prepared in a manner similar to that described in Example 2 and the signal obtained in the presence of a saturating concentration of unlabeled Notch1 / Fc. Expressed as% inhibition using the maximum inhibition defined. N.T. represents uninspected.

Structural analysis of anti-DLL4 antibody The variable heavy chain and variable light chain sequences of this antibody were analyzed to determine their DNA sequences. Complete sequence information for anti-DLL4 antibodies is presented in a sequence listing listing nucleotide and amino acid sequences for each γ and κ chain combination. This variable heavy chain sequence was analyzed to determine the VH family, D region sequence and J region sequence. This sequence was then translated to determine the primary amino acid sequence and assessed for somatic hypermutation compared to germline VH, D and J region sequences.

Table 2 is a table comparing antibody heavy chain regions to their cognate germline heavy chain regions and kappa light chain regions to their cognate germline light chain regions. The variable (V) region of an immunoglobulin chain is encoded by various germline DNA fragments that are joined to a functional variable region (V _H DJ _H or V _K J _K ) in B cell development. The molecular and genetic diversity of the antibody response to DLL4 was studied in detail.

  It should also be understood that if a particular antibody differs from its respective germline sequence at the amino acid level, the antibody sequence can be mutated back to the germline sequence. Such corrective mutations can occur at 1, 2, 3 or more positions, or any combination of mutated positions, using standard molecular biology techniques. Non-limiting examples include mutations from V to A at position 18 (mutation 1), V to A at position 32 (mutation 2), E to D at position 50 (mutation 3), S to N at position 65 (mutation 1). Table 8 shows 2H10 light chain sequences (SEQ ID NO: 6) that differ from the corresponding germline sequences by mutation 4), T to A at position 89 (mutation 5), and L to T at position 94 (mutation 6). (See Table 2). Thus, the nucleotide sequence encoding an amino acid or 2H10 light chain can be modified at any site or any site. The locations of such variations of the 2H10, 9G8, 21H3, and 4B4 germline are illustrated in Tables 2-9. Each row is bold and represents a unique combination of germline and non-germline residues at the specified position.

  In another embodiment, the invention includes the replacement of any structural disorder in the sequence that can affect the heterogeneity of the antibodies of the invention. Such obstacles include glycosylation sites, unpaired cysteines, surface exposed methionine and the like. In order to reduce such heterogeneity risk, it is proposed to change to eliminate one or more such structural obstacles.

In one example, the unpaired cysteine can be replaced alone or in conjunction with other structural changes. Examples of unpaired cysteines are expressed in the light chain CDR1 at position 33 of antibody 2H10 or 9G8. This unpaired cysteine can be mutated to an appropriate amino acid (such as serine) with similar side chain properties. In another example, unpaired cysteine is expressed in heavy chain FR4 at position 203 of antibody 20G8. This unpaired cysteine can also be mutated to an appropriate amino acid (such as serine) with similar side chain properties.

In some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 30. In certain embodiments, SEQ ID NO: 30 comprises any one combination of germline and non-germline residues shown in each row of Table 5. In some embodiments, SEQ ID NO: 30 is any one of germline residues as shown in Table 5, any two, any three, any four, any five, any 6 or all of 6 are included. In certain embodiments, SEQ ID NO: 30 comprises any one unique combination of germline and non-germline residues shown in each row of Table 5. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having VH1-18, D2-15 and JH3 regions, and one or more residues at that position corresponds to the corresponding germline. Mutated to yield residues. Specific examples of the 21H3 heavy chain variable region mutated to a specific germline sequence include 21H3 VHOP shown in Table 13 (non-germline sequence is mutated to L at position 45 and M at position 70). Are).

In some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 32. In certain embodiments, SEQ ID NO: 32 comprises any one combination of germline and non-germline residues shown in each row of Table 6. In some embodiments, SEQ ID NO: 32 is any one, any two, any three, any four, any five, or any of the germline residues as shown in Table 6. Includes all five. In another embodiment, the targeted binding agent or antibody is from a germline comprising a VL, 1g, JL2 region, and one or more residues have been mutated to provide the corresponding germline residue at that position. Specific examples of 21H3 light chain variable regions mutated to specific germline sequences include 21H3VLOP1 (optimized by mutating non-germline sequence to K at position 107) and 21H3VLOP2 (non-germline sequence , And optimized by mutation to K at positions 67 and 107). See Table 13.

In some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 2. In certain embodiments, SEQ ID NO: 2 comprises any one combination of germline and non-germline residues shown in each row of Table 7. In some embodiments, SEQ ID NO: 2 is any one of germline residues as shown in Table 7, any two, any three, any four, any five, any 6, any 7, any 8, any 9, any 10, any 11, or all 11. In certain embodiments, SEQ ID NO: 2 comprises any one unique combination of germline and non-germline residues shown in each row of Table 7. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having VH1-18, D7-27, and JH4 regions, and one or more residues at that position corresponds to the corresponding germline. Mutated to yield residues.

In some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 4. In certain embodiments, SEQ ID NO: 4 comprises any one combination of germline and non-germline residues shown in each row of Table 8. In some embodiments, SEQ ID NO: 4 is any one, any two, any three, any four, any five, or any of the germline residues as shown in Table 8. Includes all five. In certain embodiments, SEQ ID NO: 4 comprises any one unique combination of germline and non-germline residues shown in each row of Table 8. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having a VL, 3p, JL2 region, and one or more residues results in the corresponding germline residue at that position. It has been mutated.

In further forms of some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 6. In certain embodiments, SEQ ID NO: 6 comprises any one combination of germline and non-germline residues shown in each row of Table 9. In some embodiments, SEQ ID NO: 6 is any one of germline residues as shown in Table 9, any two, any three, any four, any five, any 6 or all of 6 are included. In certain embodiments, SEQ ID NO: 6 comprises any one unique combination of germline and non-germline residues shown in each row of Table 9. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having Vh3-33, D6-13, and JH4 regions, and one or more residues at that position corresponds to the corresponding germline. Mutated to yield residues. An example of a mutated sequence is 2H10 VHOP, in which M at position 93 is mutated to V. See Table 13.

In further embodiments of some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 8. In certain embodiments, SEQ ID NO: 8 comprises any one combination of germline and non-germline residues shown in each row of Table 10. In some embodiments, SEQ ID NO: 8 is any one, any two, any three, any four, any five, any, germline residues as shown in Table 10 6 or all of 6 are included. In certain embodiments, SEQ ID NO: 8 comprises any one unique combination of germline and non-germline residues shown in each row of Table 10. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having VL, 3r and JL2 regions, and one or more residues at that position results in the corresponding germline residue It has been mutated. In certain embodiments, SEQ ID NO: 8 can comprise a modification that includes removal of a structural disorder. For example, in addition to germline, C33 can be mutated to S. See for example 2H10 VLOP1 in Table 13. Thus, SEQ ID NO: 8 includes any one of the unique combinations of germline and non-germline residues shown in each row of Table 10, and may further include a C33 to S mutation. An example of a sequence that was mutated in C33 to eliminate the light chain structural disorder and then mutated back to the germline sequence is 2H10 VLOP2 shown in Table 13, C33 mutated to S, 18th position V Was mutated to A and S at position 65 was mutated to N.

In some embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 22. In certain embodiments, SEQ ID NO: 22 comprises any one combination of germline and non-germline residues shown in each row of Table 11. In some embodiments, SEQ ID NO: 22 is any one, any two, any three, any four, any five, any six, any seven shown in Table 11 Contains one or all seven germline residues. In certain embodiments, SEQ ID NO: 22 comprises any one unique combination of germline and non-germline residues shown in each row of Table 11. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having VH4-39, D4-23, and JH3 regions, wherein one or more residues have a corresponding germline residue at that position. Mutated to give a group. An example of a sequence in which the heavy chain was mutated to the original germline sequence was 9G8 VHOP1 shown in Table 13, and L at position 38 was mutated to W. Another example of the sequence in which the heavy chain was mutated to the original germline sequence is 9G8 VHOP2 shown in Table 13, where S at position 21 is mutated to T, L at position 38 is mutated to W, S was mutated to T, and F at position 96 was mutated to Y.

  In some further embodiments of the invention, the targeted binding agent or antibody comprises a sequence comprising SEQ ID NO: 8. In certain embodiments, SEQ ID NO: 24 comprises any one combination of germline and non-germline residues shown in each row of Table 12. In some embodiments, SEQ ID NO: 24 is any one, any two, any three, any four, any five, any six, any seven shown in Table 12 One, any 8, any 9, any 10, any 11, or a total of 11 germline residues. In certain embodiments, SEQ ID NO: 24 comprises any one unique combination of germline and non-germline residues shown in each row of Table 12. In other embodiments, the targeted binding agent or antibody is derived from a germline sequence having a VL, 3r, and JL2 region such that one or more residues results in the corresponding germline residue at that position. Has been mutated. In certain embodiments, SEQ ID NO: 24 can include further modifications including removing structural disturbances. For example, SEQ ID NO: 24 can comprise any one unique combination of germline and non-germline residues shown in each row of Table 12, and can further comprise a C33 to S mutation. An example is 9G8 VLOP1, which mutates the light chain to remove the structural deficiency of C33, further mutates the original germline sequence at position 7 (S is mutated to P), and mutates V at position 79 to A. It was. Another example is 9G8 VLOP2, mutating C33 to S, mutating S at position 2, mutating S to Y, mutating S at position 7 to P, mutating R at position 19 to S, mutating T at position 39 to P , 79 position V was mutated to A. See Table 13 for details.

Those skilled in the art will realize that there are other ways to define CDR boundaries. All CDR boundaries in Tables 2 and 13 are defined according to the Kabat definition.

Potency determination of DLL4 antibodies that inhibit Notch1-DLL4 receptor-ligand binding Based on the ability to prevent interaction between full-length recombinant DLL4 and soluble Notch1 / Fc, the following was used to distinguish purified antibodies: The assay was performed. Transfected and non-transfected 239T cells were reconstituted with PBS containing 2% FCS and 5000 transfected and 17500 untransfected cells were reconstituted in 384 well tissue culture plates (Corning Costar, A well of catalog # 3712) was seeded in a volume of 30 μl. Thereafter, 30 μl of purified antibody serially diluted from 5 μg / ml hybridoma supernatant was added and the plate was incubated for 1 hour at 4 ° C., at which time 20 μl of 100 ng / ml Alexa-647 labeled human Notch1 / Fc was added. . After further incubation at 4 ° C. for 3 hours, the amount of Notch1 / Fc binding was measured by reading the fluorescence of each well using an FMAT 8200 instrument (Applied Biosystems). Data for 12 purified antibodies is shown in Table 14 and shows the ability of the purified antibody to inhibit the interaction between recombinant full-length human DLL4 and Notch1 / Fc chimera.

  Furthermore, the same experiment was performed and quantified using a FACSCalibur (BD Biosciences) apparatus. For these experiments, parental 293T cells or cells transiently transfected with human DLL4 were reconstituted with PBS containing 2% FCS to a final concentration of 10 μg / ml, 1 μg / ml or 0.1 μg / ml. To wells containing purified antibody was added at a concentration of 25000 cells / well. After 1 hour incubation at 4 ° C., Alexa-647 labeled human Notch1 / Fc was added at a final concentration of 227 ng / ml and the plate was incubated at 4 ° C. for 2 hours. After washing with PBS containing 2% FCS, the Notch1 / Fc binding amount was measured by reading the fluorescence of each well using a FACSCalibur apparatus. Under these conditions, the ability of the 20 purified antibodies to inhibit DLL4-Notch1 interaction was similar to that observed using the FMAT instrument (data not shown). Similar results were obtained when experiments were performed in ELISA format using soluble human DLL4 and soluble human Notch1 (data not shown).

Further experiments were performed on selected antibodies using HEK293 cells stably transfected with either human, mouse or cynomolgus DLL4 using retroviral constructs. In these experiments, anti-DLL4 antibody (final concentration: 10-0.01 μg / ml) diluted in PBS containing 2% FCS was injected into HEK293 cells expressing DLL4 (50,000 diluted in PBS containing 2% FCS). Cells / well) and incubated at 4 ° C. for 1 hour. Alexa-647-labeled human, rat or APC-labeled mouse Notch1 / Fc (eg, R & D Systems, catalog # 3647-TK-050, 1057-TK-050, 5267-TK-050, respectively) at a final concentration of 0.01 The plate was incubated at ˜0.5 μg / ml for an additional 2 hours at 4 ° C., washed and read on a FACSCalibur instrument. Recombinant full-length human, cynomolgus monkey and mouse DLL4 as measured by FACS analysis and human, rat or 0.5 μg / ml concentration mouse Notch1 / Fc chimera at a concentration of 0.1 or 0.25 μg / ml The ability of purified antibodies of different isotopes to inhibit the interaction between is shown in Table 15.

Cross-reactivity of DLL4 antibody to human JAGGED1 and DLL1 The ability of the purified antibody to bind to human Jagged1 and Dll1 was measured by FACS analysis. Briefly, 293T cells were mock transfected using Lipofectamine 2000 or transiently using either human Jagged1 (Accession #: NM_000214) or Dll1 (Accession #: NM_005618). Transfected. Cells were resuspended in PBS containing 2% FCS and seeded in V bottom plates at 50000 cells / well. Anti-DLL4 antibody diluted in PBS containing 2% FCS was added at a final concentration of 5 or 15 μg / ml and the plate was incubated at 4 ° C. for 1 hour. After washing with PBS containing 2% FCS, a secondary antibody (goat anti-human Fc Cy5, Jackson Immunoresearch, catalog # 109-175-098, 5 μg / ml) and 7-AAD (5 μg / ml) were added at 4 ° C. Plates were incubated for 15 minutes, washed again with PBS containing 2% FCS and read on a FACSCalibur instrument. Mouse anti-human Jagged1 antibody (R & D systems, catalog # mAB1277, Jackson Immunoresearch catalog # 115-175-164, detected with secondary antibody of anti-mouse Fc Cy5 (5 μg / ml), human Notch3 / Fc chimera (R & D systems) , Catalog # 1559-NT-050, detected with the above goat anti-human Fc Cy5 antibody or goat anti-human Dll1 antibody (R & D systems cat # AF1818, detected with anti-goat Fc Cy5, Jackson Immunoresearch, catalog # 305 −175-046 (5 μg / ml)) was used as a control to confirm transfection. Data were analyzed by comparing the shift of the fluorescence mean of mock-transfected cells to the geometric mean of fluorescence of Jagged1 or Dll1 transfected cells. The data is shown in Tables 16 and 17. Table 16 shows the binding ability of purified antibody (5 μg / ml) to 293T cells transiently transfected with human Jagged1. Table 17 shows the ability of the purified antibody (15 μg / ml) to bind to 293T cells transiently transfected with human Dll1. Additional FACS binding studies using 21H3RK, 4B4, 9G8 or 2H10 at a concentration of up to 300 μg / ml showed minimal binding to HEK-293 cells stably overexpressing either human Jagged1 or human Dll1 ( <2.5 times background).

Measurement of the effect of DLL4 antibody on DLL4-mediated HUVEC proliferation The ability of DLL4 antibody to block inhibition of HUVEC proliferation by DLL4 stimulation was evaluated. The extracellular region of DLL4 (R & D systems, catalog # 1506-D4 / CF) was prepared in PBS containing 0.1% BSA as a 50 μg / ml stock. After dilution to 1 μg / ml with bicarbonate buffer (Sigma # C3041-50CAP), 100 μl / well was added to a black wall 96 well plate (Perkin Elmer, catalog # 60000512) and the plate was incubated overnight at 4 ° C. Control wells were also mock coated with PBS containing 0.1% BSA. After washing with PBS, 100 μl of HUVEC cells were added to each well at a concentration of 4E4 cells / ml in MCDB113 (Gibco catalog # 10372) containing 10% FCS and 2 mM glutamine. Immediately thereafter, serially diluted anti-DLL4 antibody (20-0.027 μg / ml) was added in triplicate to DLL4 / mock-coated wells and the cells were incubated at 37 ° C./5% CO ₂ for 96 hours. After this incubation, 15 μl of Cell Counting Kit 8 (CCK8, NBS, catalog # CK-04-11) was added to each well and the plate was incubated at 37 ° C./5% CO ₂ for 4 hours. In order to determine the relative number of cells in each well, the absorbance at 450 nm was measured with a plate reader (Tecan Ultra). The effects of anti-DLL4 antibodies are listed in Table 18. Furthermore, 4B4, 21H3 and 21H3RK were also effective inhibitors of DLL4-mediated effects when formatted as IgG1 antibodies (FIG. 1).

Furthermore, the ability of anti-DLL4 antibody (10 μg / ml) to inhibit Notch signaling was assessed by Western blot. Briefly, DLL4-His (R & D systems, catalog # 1506-D4-050 / CF) was diluted to 50 μg / ml with PBS containing 0.1% BSA. This solution was then further diluted to 1 μg / ml with 50 mM bicarbonate buffer pH 9.6 (Sigma catalog # C-3041) and 1 ml per well was added to a 12-well plate and incubated at 4 ° C. overnight. Additional wells without DLL4 were mock coated using the same method. After washing with PBS, HUVEC cells prepared in MCDB131 medium were seeded at 12000 cells / well. Immediately after seeding, the appropriate treatment (eg, anti-DLL4 antibody, 10 μg / ml) was added and the plates were incubated at 37 ° C./5% CO ₂ for 24 hours. After incubation was complete, cells were harvested with RIPA buffer. Thereafter, 4 × sample buffer containing β-mercaptoethanol and bromophenol blue was added and the sample was boiled at 70 ° C. for 5 minutes and loaded onto a 4-12% NuPAGE gel in MOPS buffer (Invitrogen catalog # NP0001). After electrophoretic transfer to nitrocellulose, the blot was blocked with PBST containing 5% milk for 1 hour and cleaved anti-Notch1 antibody (Cell) diluted 1: 1000 or 1: 10,000 respectively in PBS containing 5% milk. Incubated with either signaling technology, catalog # 2421) or GAPDH antibody (Advanced Immunochemical, clone 6C5, catalog # 2-RGM2). After incubation overnight on an orbital shaker at 4 ° C., the blot was washed with PBST and anti-mouse HRP secondary antibody (Cell) at a concentration of 1: 2000 in PBST containing 5% milk at room temperature for 1 hour. Incubated with Signaling Technology, catalog # 7072). After washing with PBST, the blots were developed using Pierce Pico (Catalog # 34080; GAPDH) or Femto (Catalog # 34075; Cleaved Notch1) substrate reagents and the results analyzed on a ChemiGenius instrument. The results obtained from these studies indicate that anti-DLL4 antibodies can block DLL4-stimulated Notch signaling in HUVEC cells (data not shown).

Effect of DLL4 antibodies on in vitro HUVEC tube formation The ability of DLL4 inhibitory antibodies to reduce endothelial cell tube formation in an in vitro co-culture assay was tested (eg, TCS Cell Works Cat no. ZHA-1000). Human umbilical vein endothelial cells (HUVEC) and human diploid fibroblasts were obtained as co-cultures in 24-well plates (TCS Cell works Cat no. ZHA-1000) or plates were prepared as follows. A 24-well tissue culture plate was coated with collagen (1:10 dilution with distilled water; Sigma, catalog # C8919) for 4 hours at 37 ° C./5% CO ₂ . After washing with PBS, fibroblasts (eg, Promocell # C-12300) were added to FGM (Promocell # C-23010) at 15000 cells / well. After 3 days incubation at 37 ° C./5% CO ₂ , the medium was removed from the plate and HUVEC cells were added at 30,000 cells / well in EGM2 (Promocell # C-22111). After an additional 4 days of incubation at 37 ° C./5% CO ₂ , the plates were considered ready for use and this was considered the first day of the assay. DLL4 inhibitory antibodies were introduced into the culture medium on day 1 and introduced periodically over 11 days at concentrations ranging from 20-0.027 μg / ml. The medium was replenished on days 4, 7 and 9. This co-culture model was developed using TCS-optimized medium (provided with the co-culture assay) or MCDB131 medium supplemented with 2% fetal calf serum (FCS), 1% glutamine and 1% penicillin / streptomycin (from now on 2% FS (Referred to as MCDB131 medium). This co-culture model was maintained at 37 ° C. in a humidified 5% CO ₂ /95% air atmosphere. Tubular formation was examined on day 11 and CD31 tubules were fixed and stained using a tubule staining kit (TCS Cell Works catalog # ZHA-1225) according to the manufacturer's instructions. Briefly, cells were fixed with ice-cold 70% ethanol for 30 minutes at room temperature (RT). Cells were blocked and then treated with anti-human CD31 for 60 minutes at RT. After washing the plate, it was treated with goat anti-mouse IgG conjugated with alkaline phosphatase (AP) for 60 minutes at RT. After incubation with the AP-conjugated secondary antibody, the plates are washed and 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT) substrate is added for approximately 10 minutes. It was. Dark purple color development within 10 minutes reflected tube formation. The plate was then washed and air dried. Quantification of tube growth was performed by whole-well image analysis methodology using a Zeiss KS400 3.0 image analyzer. The morphological parameter measured with the quantification methodology was the total length of the tube. In some experiments, the number of tube branches was also evaluated. All tube formation in each 24 well was measured excluding the 100 μm deep edges to avoid end-retraction artifacts.

As illustrated in FIG. 2, we observed that these antibodies were effective in inhibiting endothelial cell tube formation in vitro. In addition, the titer of several antibodies was measured in this assay. These data are summarized in Table 19. Taken together, this data indicates that these antibodies are active in functional assays that model angiogenic processes.

Determination of the binding affinity of the purified antibody The binding affinity of the purified antibody to DLL4 was measured using both FACS and BIAcore techniques. For FACS affinity measurement, HEK293 cells overexpressing either human or cynomolgus monkey DLL4 were seeded at approximately 85,000-104,000 cells / well and purified antibody titration (4 ° C. for 5 hours) incubation). The cells were then washed and incubated with goat anti-human IgG-Fc-Cy5 and 5 μg / mL 7-amino-actinomycin (7AAD) at 4 ° C. for 30 minutes. Bound DLL4 was detected using FACS analysis and the data was fit to the following equation (see Drake & Krakamp, 2007, J. Immunol. Methods, 318, 157-162 for derivation).

In this equation, F is fluorescence mean value, L _T is the total molecular mAb concentration, P 'is the cell concentration constant of proportionality, M is molarity to associate with mAb bound any fluorescence units, n represents number of receptors per cell , B is background signal, and _the K _D is the equilibrium dissociation constant. For each mAb titration curve, an estimate of K _D is obtained as P ′, and n, B, and K _D can be freely varied by nonlinear analysis. Table 20 summarizes the affinity estimates of anti-DLL4 antibodies for human and cynomolgus monkey DLL4 obtained using the above methodology.

For Biacore analysis, each purified anti-DLL4 antibody was immobilized on a CM5 sensor chip in T100 using standard amine linkage. The fixed amount was kept below 200 RU. Developed by Pace et al. (GR Grimsley and C.N. Pace (2003) in Current Protocols in Protein Science (John Wiley & Sons, Inc.), 3.1.1-1.9). The concentration of DLL4 was measured by UV-VIS spectroscopy using a molar extinction coefficient at 280 nm of 110,440 M ⁻¹ cm ⁻¹ calculated from the protein sequence. Antigen DLL4 (R & D systems; human catalog # 1056-D4-050 or mouse, catalog # 1389-D4-050) was diluted to a starting concentration of 32-64 nM and tested in triplicate at 3-fold serial dilutions. The running buffer contained HBS-P with 0.1 mg / ml BSA and the binding reaction was collected at 23 ° C. The bound complex was regenerated using a 15 second pulse of 10 mM sodium hydroxide. When evaluating the binding to human soluble DLL4, a simple reaction data 1: 1 generally fit the interaction model, _k a obtained for anti-DLL4 antibodies, collectively _{k d} and _{K D} estimates in Table 20 It was. In addition, affinity estimates of 21H3, 21H3RK and 4B4 to soluble mouse DLL4 were also obtained using BIAcore, and all IgG type antibodies had an affinity of 360 nM or less for soluble mouse DLL4.

Measurement of Cross-Competition for DLL4 by FACS Analysis (CROSS-COMPETION) The ability of purified DLL4 antibodies to inhibit the binding of other DLL4 antibodies to human DLL4 was evaluated using a FACS assay. Briefly, antibodies were directly labeled with Alexa-647 using a commercial labeling kit (eg, Molecular Probes catalog # A30009, A-20186) according to the manufacturer's instructions. To measure the amount of cross-competition, unlabeled anti-DLL4 antibody (final concentration: 10-0.01 μg / ml) diluted in PBS containing 2% FCS was added to HEK293 cells expressing DLL4 (described in Example 6). As above; 50000 cells / well diluted with PBS containing 2% FCS) and incubated at 4 ° C. for 1 hour. Thereafter, Alexa-647-labeled anti-DLL4 antibody was added at a final concentration of 0.1 μg / ml, the plate was further incubated at 4 ° C. for 1 hour, washed and then read on a FACSCalibur instrument. Data summarizing the ability of unlabeled antibodies (10, 1, 0.1 μg / ml) to compete with labeled 21H3RK for binding to human DLL4 is included in FIG. Additional data summarizing the ability of unlabeled antibodies to compete with labeled 21H3RK and 4B4 for binding to human DLL4 is included in Table 22.

In addition, the ability of antibodies to detect recombinant DLL4 expressed in HEK293 cells (R & D systems, catalog # 1506-D4-050 / CF) and native DLL4 was also measured by Western blot using standard techniques. (See Example 3). Briefly, HEK293 cells expressing DLL4 are harvested in RIPA buffer (Thermo, catalog # 89900) containing one protease inhibitor mixed tablet (Roche, catalog # 11873580001) and used by the manufacturer. The protein was quantified using the BCA protein assay (Pierce, catalog # 23227) according to the instructions. For Western blots, protein samples are thawed on ice, incubated for 2 minutes at 100 ° C., 10 × Nupage sample reducing agent (Invitrogen, catalog # NP0004) and 4 × sample buffer are added, MES buffer (Invitrogen, catalog # NP0002) Medium precast 4-12% NuPage BisTris gel (Invitrogen, catalog # NP0321B0X). After electrophoretic transfer to nitrocellulose, blots were added to a solution of 0.05% Tween 20 in Tris buffered saline (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) (TBST) plus 5% milk. 9G8, 2H10, 21H3, 4B4 or 20G8 (all 2 μg / ml) or commercially available anti-DLL4 antibody (R & D Systems, catalog # MAB1389; Abcam, catalog # ab7280, in TBST containing 5% milk. Both were incubated with either 1 μg / ml). After incubation overnight on an orbital shaker at 4 ° C., the blot was washed with TBST and anti-rat, anti-rabbit or anti-human HRP-conjugated secondary antibody in TBST containing 5% milk at room temperature for 1 hour. (Jackson Immunochemicals, catalog # 112-035-003 and 111-035-003, 1: 20,000 dilution or KPL, catalog # 074-1006, 1: 10,000 dilution). Excess antibody is removed by washing as described above, and immune complexes are visualized by enhanced chemiluminescence detection according to manufacturer's instructions (Pierce, catalog # 34076) and Hyperfilm ECL (Amersham, catalog # 28906839) or Biomax Detected on MR (Kodak, catalog # 8952855) film. Under these conditions, both recombinant and native DLL4 can be detected by commercially available antibodies and 9G8 / 2H10, but these results indicate that they cannot be detected by 21H3 / 20G8 or 4B4, and these antibodies are detected on DLL4 Suggests that they may interact with different epitopes (data not shown).

Sequence modification of 9G8 and 2H10 During the immune response maturation period, immunoglobulin genes undergo various modifications, including recombination between variable region V, D and J gene fragments, isotype switching, and hypermutation. Recombination and somatic hypermutation are the basis for the generation of antibody diversity and affinity maturation, but can also result in sequence defects that can make industrial production of such immunoglobulins as therapeutic agents difficult Or may increase the immunogenicity risk of the antibody. In general, mutations in the CDR regions are likely to contribute to improved affinity and function, but mutations in the framework regions may increase the immunogenicity risk. This risk can be reduced by returning framework mutations to the germline, ensuring that the activity of the antibody is not adversely affected. Some structural disorders can be born by diversification processes or can be present in germline sequences that contribute to the heavy and light chain variable regions. Regardless of the source, it may be desirable to remove potential structural disturbances that can result in instability, aggregation, product heterogeneity, or increased immunogenicity. Examples of undesirable obstacles include unpaired cysteine (which can result in disulfide bond scrambling, or variable sulfhydryl adduct formation), N-linked glycosylation sites (which result in heterogeneity of structure and activity), and deamidation (Eg, NG, NS), isomerization (DG), oxidation (exposed methionine) and hydrolysis (DP) sites. In an attempt to reduce the risk of immunogenicity and improve the pharmaceutical properties of the lead antibody, specific variants were generated and tested for binding and activity. Site-directed mutagenesis was performed using the Stratagene Quick Change II kit as described by the manufacturer. Variant sequences were expressed by Invitrogen Freestyle system by transient transfection (according to the manufacturer's recommended procedure) and purified by protein A affinity chromatography.

Mutant antibody activity was assessed by two methods: first, blocking soluble Notch1 / Fc binding to full-length human DLL4 stably expressed in HEK293 cells as described in Example 3 The ability of the antibody was measured and secondly the binding of the antibody to the same cell line used in the receptor-ligand competition test. For binding studies, HEK293 cells stably overexpressing human DLL4 were resuspended in PBS containing 2% FCS at a concentration of 50,000 cells / well and antibody tight for 1 hour at 4 ° C. Incubation (final concentration 0.01-10 μg / ml). The cells were then washed and incubated with 0.31 μg / ml anti-human IgG-Fc-FITC (BD Pharmingen, cat # 555786) for 30 minutes at 4 ° C. Bound DLL4 was detected using FACS analysis. Results of these studies showing the effect of specific mutations in the sequences of 9G8 and 2H10 on competition with Notch1 in binding to human DLL4 compared to unmutated antibodies, or the ability of these antibodies for binding to human DLL4 Are summarized in Table 23.

Evaluation of the efficacy of angiogenesis inhibition in spheroid-based in vivo angiogenesis assays Human umbilical vein endothelial cells (HUVEC) spheroids were prepared as previously described (Korff and Augustin: J. Cell. Biol. 143: 1341-). 52, 1998), 100 endothelial cells (EC) were prepared by hanging by pipetting on a plastic dish in a hanging drop method and allowing the spheroids to form overnight. The next day, the EC spheroids were collected using the method described previously (Aladati et al., Nature Methods 5: 439-445, 2008) and mixed into a Matrigel / fibrin solution with a single HUVEC and finally Typical numbers reached 100,000 EC as spheroids and 200,000 single EC per injected plug. VEGF-A and FGF-2 were added at a final concentration of 1000 ng / ml. 500 μl of cell suspension per matrix was injected subcutaneously into male SCID mice (5-8 weeks old). The next day (Day 1) began treatment. On the 21st day, the study was terminated. The matrix plug was removed and fixed with 4% PFA. For histological examination, all matrix plugs were embedded in paraffin and cut to a thickness of 8-10 μm. Vessels were visualized by staining with human CD34 and smooth muscle actin (SMA), and vessel density and pericyte coverage were measured. Both IgG1 and IgG2 antibodies are effective in regulating in vivo angiogenesis and inhibiting pericyte mobilization. The data obtained shows that treatment with anti-DLL4 antibody (21H3RK or 4B4) induces an increase in human angiogenesis of at least 100% over untreated controls at antibody concentrations as low as 1 mg / kg. I suggest that. Furthermore, an increase in human angiogenesis is associated with a decrease in pericyte cell coverage of at least 50% (as assessed by the percentage of human CD34 positive vessels also associated with αSMA-expressing positive cells) at an antibody dose of 5 mg / kg. did. Table 24 shows data summarizing the effects of 21H3RK IgG1 administered intraperitoneally twice weekly at concentrations of 1, 0.2 and 0.04 mg / kg. Taken together, this data indicates that these antibodies are active in in vivo assays of angiogenesis.

Epitope mapping of 21H3RK Monoclonal 21H3RK specifically binds to human DLL4 but does not recognize human DLL1. This specificity is used to estimate the binding epitope of Mab 21H3RK for human DLL4. The chimeric variant was genetically engineered to replace the portion of the extracellular region of DLL4 with the corresponding portion of DLL1. To construct a series of variants containing DLL4 transmembrane regions for surface expression of recombinant proteins (Yoneya et al., 2001, J. Biochem., 129, 27-34, cloned in the laboratory) Human DLL4 and DLL1 (Accession # NM_005618, Origen, MD) were used as templates for the overlap extension PCR method. The resulting variant was cloned into a mammalian expression vector encoding human cytomegalovirus major immediate early (hCMVie) enhancer, promoter and 5'-untranslated region for transient mammalian expression. For characterization of Mab 21H3RK by flow cytometry, the chimeric variant was transiently expressed in HEK293F cells as a membrane bound protein. 48 hours after transfection, HEK293F transfectants were incubated with 1 μg / ml Mab 21H3RK in PBS for 1 hour on ice, washed, and then goat anti-human IgG-FITC (Jackson ImmunoResearch Laboratories, PA). Incubated and analyzed using an LSRII flow cytometer (BD Biosciences, CA). The expression level of all chimeric variants was measured by incubating with a mixture of both goat anti-mouse DLL4 (which also recognizes human DLL4) and goat anti-human DLL1 polyclonal antibody (both R & D Systems., MN), Detection was with porcine anti-goat IgG-PE (Invitrogen, CA).

  Human Dll1 and Dll4 share 53% identity at the amino acid level, but Mab 21H3RK specifically binds Dll4 and does not recognize Dll1. A chimeric variant of human Dll4 encoding a small portion of human Dll1 was constructed to identify regions involved in this specificity. Twelve chimeric knockout variants were constructed by replacing the subdomain of human Dll4 with the corresponding residue of human Dll1. FIG. 4 shows how the extracellular part of DLL4 is divided into structurally defined subdomains. The large amino terminus (N-ter) of the mature DLL4 protein was divided into two small parts. KO variant N-ter1 replaces the first 86 amino acids (AA) of mature DLL4 protein with human DLL1, and KO variant N-ter2 replaces amino acids 87-146 with human DLL1. The other knockout variants shown in the figure are the entire N-terminal region (AA1-146), DSL region (AA147-191), EGF1 region (AA192-224), both EGF1 and 2 regions (AA192-255), EGF3 And 4 regions (AA256-333), and 4 EGF5-8 regions (AA334-503) Dll1 substitutions. In addition, both the N-terminal and DSL regions (AA1-191), the N-terminal and DSL and EGF1-2 regions (AA1-255), the DSL and EGF1-2 regions (AA147-255), and the DSL and EGF1 regions (AA147- Region replacement variants combining 224) were made by genetic manipulation. While all recombinant proteins were well expressed on the cell surface as measured using anti-Dll4 and Dll1 polyclonal antibodies (FIG. 5, upper panel in both rows), Mab 21H3RK is a DSL and EGF1 human Dll1 None of the constructs containing both of the regions were recognized (Figure 5, lower panel). Further, in the Dll1 construct (knock-in mutant) encoding the Dll4 DSL and EGF1 regions, Mab 21H3RK can bind to Dll1 (FIG. 6). Thus, the 21H3RK binding epitope is located within the DSL and EGF1 regions.

  Three additional variants were engineered to further refine the binding epitope in this part of the protein and identify the essential residues involved in the binding specificity of Mab 21H3RK. The three 15 amino acid parts in the DSL and / or EGF1 region of Dll4, corresponding Dll1 residues that encode a few amino acid substitutions in which the Dll1 and Dll4 sequences are not conserved: fragment A (AA187-201), fragment Replaced with B (AA200-214) and fragment C (AA210-214). Fragment A spans the last 5 amino acids of the DSL region, a 4 amino acid linker between the DSL and EGF1 regions, and 6 amino acids of the EGF1 region. Substitution of these 15 amino acids with Dll1 residue resulted in loss of Mab 21H3RK binding. When the Dll4 residue in fragments B and C was replaced with Dll1, no effect was observed. These data identified 15 amino acids (AA187-201) including the C-terminus of DSL and the N-terminus of EGF1 as important for binding, and epitopes for 21H3RK were identified as the C-terminus of DSL and the N-terminus of EGF1. Maps to the DSL and EGF1 regions (AA147-224) with the essential regions localized in (AA187-201).

Measurement of DLL4 antibody causing internalization of DLL4 by FACS analysis The ability of the purified antibody to induce DLL4 internalization was examined by FACS analysis. HEK293 cells stably overexpressing DLL4 were separated and washed with FACS buffer (PBS + 2% FCS), and seeded at 50,000 to 100,000 cells per well in a V-bottom plate. Primary antibody (anti-DLL4 or appropriate isotype control) is diluted to a final concentration of 10 μg / ml in warm 37 ° C. FACS buffer and is 30, 60, 120 in a tissue culture incubator (37 ° C./5% CO ₂ ). Or added to these cells for 240 minutes. At appropriate time points, cells were pelleted at 500 g in a centrifuge previously chilled to 4 ° C., washed with cold FACS buffer and FITC-labeled anti-human IgG secondary antibody (1 μg / ml, Jackson Labs) on ice for 10 minutes. , cat # 109-096-098). After incubation, the cells were reprecipitated at 500 g with a pre-chilled centrifuge, washed with cold FACS buffer and fixed with 2% paraformaldehyde for 20 minutes. Internalization was assessed by reading with a FACSCalibur. Under these assay conditions, <10% internalization of 21H3RK occurred at the time points described above. Internalization can also be measured by incubating with antibodies against DLL4 that do not cross-compete instead of secondary anti-human IgG antibodies. In some experiments, primary antibody (10 μg / ml) diluted in FACS buffer is pre-incubated with cells overexpressing DLL4 for 30 minutes on ice as described above and washed with warm 37 ° C. FACS buffer. Cells were incubated for 30, 60, 120 or 240 minutes in a tissue culture incubator (37 ° C./5% CO ₂ ). Following these incubations, the cells were washed, incubated with secondary antibody, fixed as described above, and read on a FACSCalibur. Under these conditions, <15% of 21H3RK and <35% of 4B4 were observed at 60 minutes and 240 minutes, respectively, relative to the 0 minute control. Under the assay conditions described above, internalization can also be measured by incubating with an antibody against DLL4 that does not cross-compete instead of a secondary anti-human IgG antibody.

Activity of anti-DLL4 antibodies in a mouse Matrigel plug model of angiogenesis The ability of anti-DLL4 antibodies to modulate angiogenesis can be assessed using a Matrigel plug assay. In this assay, compounds that induce angiogenesis, such as bFGF, VEGF, or tumor cells, can be introduced into liquid Matrigel, which clots after subcutaneous injection and is infiltrated by cells of endothelium and vascular smooth muscle. Allow new blood vessels to form. Briefly, at 4 ° C., liquid matrigel is mixed with vehicle (eg, PBS) or growth factor / tumor cells (eg, LL2, MCF7, A431, Colo205, KRRK, Calu-6, SW620, Pancl). 0.5 ml can be injected subcutaneously into the lower abdomen of female 129s1 / SvlmJ mice (6-8 weeks old, 5 per group). Anti-DLL4 antibody can be administered twice a week by intraperitoneal injection. After 5-10 days, animals are humanely euthanized for assessment of angiogenesis, plugs are collected, eg, immunostaining for CD31 and α-smooth muscle actin (αSMA), measurement of hemoglobin content, and eg FITC -Angiogenesis can be measured by histological scoring of vessel density and wall cell coverage by evaluating vascular perfusion measurements using dextran. In this way, the ability of anti-DLL4 antibodies to modulate angiogenesis is measured.

Anti-DLL4 Antibody Activity in Human Tumor Xenograft Models Derived from Primary Patient Tumor Samples This example inhibits or prevents tumor growth from primary patient samples when grown as xenografts in mice To use anti-DLL4 antibodies for this purpose. Briefly, recipient mice can be anesthetized by isoflurane inhalation until the amount of anesthesia required for surgery is reached. The primary tumor is then rinsed with RPMI supplemented with antibiotics and 10% FCSi and minced with a scalpel to produce a “mix of muddy” and divided into appropriate transplant doses (eg, 4 mg of 300 mg tumor). Can be transplanted into one mouse). The tumor mixture can be loaded into a 13 gauge cancer implantation trocar. The shaft of the trocar can be completely filled with the tumor mixture, injected subcutaneously into the right flank, and its contents can be dispensed under the back fat pad. The mouse can then be returned to the cage and recovery monitored.

At the first passage, generally 3-5 mice are transplanted with the primary tumor mixture. When tumors reached 800～1000Mm ^3, it was sliced into approximately 3 × 3 × 3 mm pieces, using a 1 fragments each mouse secondary passaged five mice. The remaining tumor material is stored in Recovery ™ cell culture freezing medium (Gibco, catalog # 12648-010) in addition to H & E staining and DNA / RNA extraction. Tumors that exceed 2 passages can be used for transplantation for efficacy studies. In an efficacy experiment, one tumor fragment is transplanted into each animal.

After tumor growth, two vertical diameters are measured. Tumor measurements and body weight can be recorded twice a week for 2 weeks after the start of treatment. The formula for calculating tumor volume is as follows: (L × W ² ) / 2.

The DLL4 antagonist antibody can be administered as a solution. Treatment can begin when the average tumor volume reaches about 100-200 mm ³ or simultaneously with tumor implantation. The treatment period can consist of a total of 28 days. DLL4 antagonistic antibodies can be administered, for example, 5, 10, or 20 mg / kg / day (ip, qd, 2 × / wk) alone or in combination with other agents. Tumor measurements and body weight are recorded twice a week for 4 weeks after initiation of treatment. Therefore, the ability of DLL4 antibodies to inhibit the growth of tumor xenografts from patient samples, either by DLL4 antibodies alone or in combination, is measured.

Claims (36)

Specifically binds to delta-like ligand 4 (DLL4),
(A) variable heavy chain (VH) complementarity determining region (CDR) 1 comprising the amino acid sequence of NYGIT (amino acid residues 31 to 35 of SEQ ID NO: 30), WISAYNGNTNYAQKLQD (amino acid residues 50 to 66 of SEQ ID NO: 30) VHCDR2 comprising the amino acid sequence, and VHCDR3 comprising the amino acid sequence of DRVPRIPVTTEAFDI (amino acid residues 99 to 113 of SEQ ID NO: 30); and
(B) variable light chain (VL) CDR1, comprising the amino acid sequence of SGSSSNIGSYFVY (amino acid residues 23-35 of SEQ ID NO: 32), VLCDR2, comprising the amino acid sequence of RNNQRPS (amino acid residues 51-57 of SEQ ID NO: 32), and VLCDR3 comprising the amino acid sequence of AAWDDSLSGHWV (amino acid residues 90 to 101 of SEQ ID NO: 32)
An isolated antibody or binding fragment thereof. 2. The antibody or binding fragment thereof according to claim 1, wherein the antibody is a fully human monoclonal antibody or a binding fragment of a fully human antibody. The antibody or a binding fragment thereof is Fab, Fab ', F (ab') ₂ The antibody or the binding fragment thereof according to claim 1, which is Fv, dAb. A composition comprising the antibody or fragment thereof according to claim 1. A pharmaceutical composition comprising the antibody or fragment thereof according to claim 1. Specifically binds to delta-like ligand 4 (DLL4),
A variable heavy chain (VH) sequence comprising an amino acid sequence of SEQ ID NO: 30, or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 30 by including 6 substitutions in any row of Table 5; and
The variable light chain (VL) sequence comprising the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 32 by including 5 substitutions in any row of Table 6
An isolated antibody or binding fragment thereof. The antibody or binding fragment thereof according to claim 6, wherein the VH sequence comprises the amino acid sequence shown in SEQ ID NO: 30 or SEQ ID NO: 75. The antibody or binding fragment thereof according to claim 6, wherein the VL sequence comprises the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 50 or SEQ ID NO: 76. The antibody or binding fragment thereof according to claim 8, wherein the VL sequence comprises the amino acid sequence shown in SEQ ID NO: 32. The antibody or binding fragment thereof according to claim 8, wherein the VL sequence comprises the amino acid sequence shown in SEQ ID NO: 50. The antibody or binding fragment thereof according to claim 7, wherein the VH sequence comprises the amino acid sequence shown in SEQ ID NO: 30. The antibody or binding fragment thereof according to claim 11, wherein the VL sequence comprises the amino acid sequence shown in SEQ ID NO: 32. The antibody or the binding fragment thereof according to claim 12, wherein the antibody is a fully human monoclonal antibody or a binding fragment of a fully human monoclonal antibody . 13. The antibody or binding fragment thereof according to claim 12, wherein the antibody or binding fragment thereof is Fab, Fab ′, F (ab ′) ₂ , Fv or dAb . A composition comprising the antibody or fragment thereof according to claim 12. A pharmaceutical composition comprising the antibody or fragment thereof according to claim 12. The antibody or binding fragment thereof according to claim 11, wherein the VL sequence comprises the amino acid sequence shown in SEQ ID NO: 50. 18. The antibody or binding fragment thereof according to claim 17, wherein the antibody is a fully human monoclonal antibody or a binding fragment of a fully human monoclonal antibody. The antibody or a binding fragment thereof is Fab, Fab ', F (ab') ₂ The antibody or the binding fragment thereof according to claim 17, which is Fv, dAb. A composition comprising the antibody or fragment thereof according to claim 17. A pharmaceutical composition comprising the antibody or fragment thereof according to claim 17. A composition for treating a malignant tumor in an animal comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to delta-like ligand 4 (DLL4), wherein the antibody or antigen-binding fragment thereof is (A) a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 30; and (b) a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 of SEQ ID NO: 32 or SEQ ID NO: The above composition comprising a VL comprising 50 CDR1, CDR2 and CDR3. 24. The composition of claim 22, wherein the animal is a human. The malignant tumor is melanoma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocyte (liver) cancer, thyroid tumor, stomach cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, glial bud Tumor, endometrial cancer, kidney cancer, colon cancer, pancreatic cancer, esophageal cancer, head and neck cancer, mesothelioma, sarcoma, biliary cancer (cholangiocarcinoma), small intestinal adenocarcinoma, childhood malignant tumor and 23. The composition of claim 22, wherein the composition is selected from the group consisting of squamous cell carcinoma. 23. The antibody or antigen-binding fragment thereof comprises (a) a VH comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 30; and (b) a VL comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 32. The composition as described. 23. The antibody or antigen-binding fragment thereof comprises (a) a VH comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 30; and (b) a VL comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 50. The composition as described. An isolated nucleic acid molecule encoding an antibody or binding fragment thereof that specifically binds to DLL4, wherein the antibody:
(A) the heavy chain variable region (VH) CDR1, CDR2, and CDR3 of SEQ ID NO: 30; and
(B) Light chain variable region (VL) of SEQ ID NO: 32 CDR1, CDR2 and CDR3
A nucleic acid molecule as described above. 28. The nucleic acid molecule of claim 27, wherein the antibody or binding fragment thereof comprises the VH amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 75. 28. The nucleic acid molecule of claim 27, wherein the antibody or binding fragment thereof comprises the VL amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 50 or SEQ ID NO: 76. 30. The nucleic acid molecule of claim 28, wherein the antibody or binding fragment thereof comprises the VH amino acid sequence set forth in SEQ ID NO: 30. 30. The nucleic acid molecule of claim 29, wherein the antibody or binding fragment thereof comprises the VL amino acid sequence set forth in SEQ ID NO: 32. 30. The nucleic acid molecule of claim 29, wherein the antibody or binding fragment thereof comprises the VL amino acid sequence set forth in SEQ ID NO: 50. 32. The nucleic acid molecule of claim 30, wherein the antibody or binding fragment thereof comprises the VL amino acid sequence set forth in SEQ ID NO: 32. 32. The nucleic acid molecule of claim 30, wherein the antibody or binding fragment thereof comprises the VL amino acid sequence set forth in SEQ ID NO: 50. 28. The nucleic acid molecule of claim 27, wherein VH is encoded by the nucleotide sequence set forth in SEQ ID NO: 29 and VL is encoded by the nucleotide sequence set forth in SEQ ID NO: 49. 28. The nucleic acid molecule of claim 27, wherein VH is encoded by the nucleotide sequence set forth in SEQ ID NO: 29 and VL is encoded by the nucleotide sequence set forth in SEQ ID NO: 31.

Images